Methods and compositions for modification of a HLA locus

ABSTRACT

Disclosed herein are methods and compositions for modulating the expression of a HLA locus or for selectively deleting or manipulating a HLA locus or HLA regulator.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 14/589,632, filed Jan. 5, 2015, which is a continuation of U.S.application Ser. No. 13/188,417, filed Jul. 21, 2011, issued on Feb. 3,2015 as U.S. Pat. No. 8,945,868, which claims the benefit of U.S.Provisional Application Nos. 61/400,009, filed Jul. 21, 2010 and U.S.Provisional Application No. 61/404,685, filed Oct. 6, 2010, thedisclosures of which are hereby incorporated by reference in theirentireties.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable.

TECHNICAL FIELD

The present disclosure is in the fields of gene expression, genomeengineering and gene therapy.

BACKGROUND

MHC antigens were first characterized as proteins that played a majorrole in transplantation reactions. Rejection is mediated by T cellsreacting to the histocompatibility antigens on the surface of implantedtissues, and the largest group of these antigens is the majorhistocompatibility antigens (MHC). These proteins are expressed on thesurface of all higher vertebrates and are called H-2 antigens in mice(for histocompatibility-2 antigens) and HLA antigens (for humanleukocyte antigens) in human cells.

The MHC proteins serve a vital role in T cell stimulation. Antigenpresenting cells (often dendritic cells) display peptides that are thedegradation products of foreign proteins on the cell surface on the MHC.In the presence of a co-stimulatory signal, the T cell becomesactivated, and will act on a target cell that also displays that samepeptide/MHC complex. For example, a stimulated T helper cell will targeta macrophage displaying an antigen in conjunction with its MHC, or acytotoxic T cell (CTL) will act on a virally infected cell displayingforeign viral peptides.

MHC proteins are of two classes, I and II. The class I MHC proteins areheterodimers of two proteins, the α chain, which is a transmembraneprotein encoded by the MHC1 gene, and the β2 microblogulin chain, whichis a small extracellular protein that is encoded by a gene that does notlie within the MHC gene cluster. The a chain folds into three globulardomains and when the β2 microglobulin chain is associated, the globularstructure complex is similar to an antibody complex. The foreignpeptides are presented on the two most N-terminal domains which are alsothe most variable. Class II MHC proteins are also heterodimers, but theheterodimers comprise two transmembrane proteins encoded by genes withinthe MHC complex. The class I MHC:antigen complex interacts withcytotoxic T cells while the class II MHC presents antigens to helper Tcells. In addition, class I MHC proteins tend to be expressed in nearlyall nucleated cells and platelets (and red blood cells in mice) whileclass II MHC protein are more selectively expressed. Typically, class IIMHC proteins are expressed on B cells, some macrophage and monocytes,Langerhans cells, and dendritic cells.

The class I HLA gene cluster in humans comprises three major loci, B, Cand A, as well as several minor loci. The class II HLA cluster alsocomprises three major loci, DP, DQ and DR, and both the class I andclass II gene clusters are polymorphic, in that there are severaldifferent alleles of both the class I and II genes within thepopulation. There are also several accessory proteins that play a rolein HLA functioning as well. The Tap1 and Tap2 subunits are parts of theTAP transporter complex that is essential in loading peptide antigens onto the class I HLA complexes, and the LMP2 and LMP7 proteosome subunitsplay roles in the proteolytic degradation of antigens into peptides fordisplay on the HLA. Reduction in LMP7 has been shown to reduce theamount of MHC class I at the cell surface, perhaps through a lack ofstabilization (see Fehling et al (1999) Science 265:1234-1237). Inaddition to TAP and LMP, there is the tapasin gene, whose product formsa bridge between the TAP complex and the HLA class I chains and enhancespeptide loading. Reduction in tapasin results in cells with impaired MHCclass I assembly, reduced cell surface expression of the MHC class I andimpaired immune responses (see Grandea et al (2000) Immunity vol13:213-222 and Garbi et al (2000) Nat Immunol 1:234-238).

Regulation of class I expression is generally at the transcriptionallevel, and several stimuli such as viral infection etc. can cause achange in transcription. The class I genes are down-regulated in somespecific tissues, and the source of this down-regulation seems to bewithin the promoter and 3′ intergenic sequences (see Cohen et al (2009)PLos ONE 4(8): e6748). There is also evidence that microRNAs are capableof regulating some class I MHC genes (see Zhu et al, (2010) Am. J.Obstet Gynecol 202(6): 592).

Regulation of class II MHC expression is dependent upon the activity ofthe MHCII enhanceosome complex. The enhanceosome components (one of themost highly studied components of the enhanceosome complex is the RFX5gene product (see Villard et al (2000) MCB 20(10): 3364-3376)) arenearly universally expressed and expression of these components does notseem to control the tissue specific expression of MHC class II genes ortheir IFN-γ induced up-regulation. Instead, it appears that a proteinknown as CIITA (class II transactivator) which is a non-DNA bindingprotein, serves as a master control factor for MCHII expression. Incontrast to the other enhanceosome members, CIITA does exhibit tissuespecific expression, is up-regulated by IFN-γ, and has been shown to beinhibited by several bacteria and viruses which can cause a downregulation of MHC class II expression (thought to be part of a bacterialattempt to evade immune surveillance (see LeibundGut-Landmann et al(2004) Eur. J. Immunol 34:1513-1525)).

Regulation of the class I or II genes can be disrupted in the presenceof some tumors and such disruption can have consequences on theprognosis of the patients. For example, in some melanomas, an observedreduction in Tap 1, Tap 2 and HLA class I antigens was found to be morecommon in metastatic melanomas (P<0.05) than in primary tumors (seeKageshita et al (1999) Am Jour of Pathol 154(3):745-754).

In humans, susceptibility to several diseases is suspected to be tied toHLA haplotype. These diseases include Addison's disease, ankylosingspondylitis, Behçet's disease, Buerger's disease, celiac disease,chronic active hepatitis, Graves' disease, juvenile rheumatoidarthritis, psoriasis, psoriatic arthritis, rheumatoid arthritis, Sjögrensyndrome, and lupus erythematosus, among others.

HLA haplotype also plays a major role in transplant rejection. The acutephase of transplant rejection can occur within about 1-3 weeks andusually involves the action of host T lymphocytes on donor tissues dueto sensitization of the host system to the donor class I and class IIHLA molecules. In most cases, the triggering antigens are the class IHLAs. For best success, donors are typed for HLA haplotype and matchedto the patient recipient as completely as possible. But donation evenbetween family members, which can share a high percentage of HLAhaplotype identity, is still often not successful. Thus, in order topreserve the graft tissue within the recipient, the patient often mustbe subjected to profound immunosuppressive therapy to prevent rejection.Such therapy can lead to complications and significant morbidities dueto opportunistic infections that the patient may have difficultyovercoming.

Cell therapy is a specialized type of transplant wherein cells of acertain type (e.g. T cells reactive to a tumor antigen or B cells) aregiven to a recipient. Cell therapy can be done with cells that areeither autologous (derived from the recipient) or allogenic (derivedfrom a donor) and the cells may be immature cells such as stem cells, orcompletely mature and functional cells such as T cells. In fact, in somediseases such certain cancers, T cells may be manipulated ex vivo toincrease their avidity for certain tumor antigens, expanded and thenintroduced into the patient suffering from that cancer type in anattempt to eradicate the tumor. This is particularly useful when theendogenous T cell response is suppressed by the tumor itself. However,the same caveats apply for cell therapy as apply for more well knownsolid organ transplants in regards to rejection. Donor T cells expressclass I HLA antigens and thus are capable of eliciting a rejectionresponse from the recipient's endogenous immune system.

Thus, there remains a need for compositions and methods for themanipulation of HLA genes and gene expression in cells.

SUMMARY

Disclosed herein are methods and compositions for manipulating HLA genecomplexes or HLA gene expression. In particular, provided herein aremethods and compositions for modulating expression of an HLA gene so asto treat HLA-related disorders, for example human disorders related toHLA haplotype of the individual. Additionally, provided herein aremethods and compositions for deleting or repressing an HLA gene toproduce an HLA null cell, cell fragment (e.g. platelet), tissue or wholeorganism. Additionally, these methods and compositions may be used tocreate a cell, cell fragment, tissue or organism that is null for justone HLA gene, or more than one HLA gene, or is completely null for allHLA genes. In certain embodiments, the HLA null cells or tissues arehuman cells or tissues that are advantageous for use in transplants.

Thus, in one aspect, engineered DNA-binding domains (e.g., zinc fingerproteins or TALE DNA binding domain proteins) that modulate expressionof an HLA allele are provided. In certain embodiments, the DNA bindingdomain comprises engineered zinc finger proteins that are non-naturallyoccurring as well as zinc finger proteins whose recognition helices havebeen altered (e.g., by selection and/or rational design) to bind to apre-selected target site. Any of the zinc finger proteins describedherein may include 1, 2, 3, 4, 5, 6 or more zinc fingers, each zincfinger having a recognition helix that binds to a target subsite in theselected sequence(s) (e.g., gene(s)). In some embodiments, one or moreof the recognition helices of the zinc finger domains of the zinc fingerprotein is non-naturally occurring. In certain embodiments, the zincfinger proteins have the recognition helices shown in Table 1. In otherembodiments, the zinc finger proteins bind to the target sites shown inTable 2. In other embodiments, the DNA binding domain comprises a TALEDNA binding domain (e.g., a TALE DNA binding domain comprising naturallyoccurring and/or non-naturally occurring TALE binding domains).

In certain embodiments, the DNA-binding proteins (e.g., zinc fingerproteins (ZFPs) or TALE DNA binding proteins) as described herein can beplaced in operative linkage with a regulatory domain (or functionaldomain) as part of a fusion protein. In certain embodiments, theregulatory domain is an activation domain or repression domain forfusion with the DNA-binding domain, and such fusion proteins (i.e. ZFP-or TALE-transcription factor fusions: ZFP-TF and TALE-TF respectively)can be used either to activate or to repress gene expression. In someembodiments, repressors are provided which are capable of preferentiallybinding to HLA genes or modulators of HLA gene expression. In certainembodiments, the activity of the regulatory domain is regulated by anexogenous small molecule or ligand such that interaction with the cell'stranscription machinery will not take place in the absence of theexogenous ligand. Such external ligands control the degree ofinteraction of the ZFP-TF or TALE-TF with the transcription machinery.

In one embodiment, the repressors are capable of binding to a specificHLA A, HLA B or HLA C transcriptional regulatory region and are able torepress expression in only one of the above genes. In another aspect, arepressor is provided that is capable of interacting withtranscriptional regulatory regions common to HLA A, HLA B and HLA C suchthat all three genes are regulated with one DNA binding domain (e.g.,ZFP TF or TALE TF). In another embodiment, a repressor is capable ofbinding to a regulator of HLA class II expression or function (e.g.CIITA or RFX5) to repress its activity and thus repress HLA class IIexpression or function.

In other embodiments, repressing DNA binding domain-transcription factorfusions are provided which preferentially bind to known HLA haplotypesto repress expression of only one allele.

In another aspect, DNA-binding domain-transcription factor fusions thatspecifically activate the expression of HLA genes are provided. Suchfusions may up-regulate a class of HLA genes by increasing expression ofa regulator, or may cause expression of such a class in tissues wherethese genes are not normally expressed. In another embodiment, providedare DNA-binding domain-transcription factor fusions (e.g., ZFP TFs orTALE TFs) that activate specific HLA genes as desired.

In another aspect, the fusion protein comprises a DNA-binding protein(e.g., ZFP or TALE) as described herein in operative linkage with afunctional domain comprising a nuclease (e.g., ZFNs or TALENs). Incertain embodiments, provided herein are zinc finger nucleases (ZFNs) orTALE DNA binding domains fused to a nuclease (TALENS) that cleave an HLAgene. In certain embodiments, the ZFNs and/or TALENs bind to targetsites in a human HLA class I gene and/or target sites in a human HLAclass II gene. In some embodiments, cleavage within the HLA gene(s) withthese nucleases results in permanent disruption (e.g., mutation) of theHLA gene. In certain embodiments, two pairs of ZFNs and/or TALENs may beused to cause larger deletions. The deletions may comprise a smallportion of one HLA gene, or HLA regulator gene, or may comprise largersegments. In some embodiments, the deletions caused by the ZFNs orTALENs may delete one or more HLA genes or may delete an entire HLA genecomplex (i.e., all of the class I HLA genes, or all of the HLA class IIgenes). The deletions may also encompass the deletion of a subset of aclass of HLA genes. The zinc finger DNA binding proteins may include 1,2, 3, 4, 5, 6 or more zinc fingers, each zinc finger having arecognition helix that binds to a target subsite in the target gene. Incertain embodiments, the zinc finger proteins comprise 4 or 5 or 6fingers (where the fingers are designated F1, F2, F3, F4, F5 and F6 andordered F1 to F4 or F5 or F6 from N-terminus to C-terminus) and thefingers comprise the amino acid sequence of the recognition regionsshown in Table 1.

Any of the ZFN or TALEN proteins described herein may further comprise acleavage domain and/or a cleavage half-domain (e.g., a wild-type orengineered FokI cleavage half-domain). Thus, in any of the ZFNs orTALENs described herein, the nuclease domain may comprise a wild-typenuclease domain or nuclease half-domain (e.g., a FokI cleavage halfdomain). In other embodiments, the ZFNs or TALENs comprise engineered(non-naturally occurring) nuclease domains or half-domains, for exampleengineered FokI cleavage half domains that form obligate heterodimers.See, e.g., U.S. Patent Publication No. 20080131962.

In another aspect, the disclosure provides a polynucleotide encoding anyof the proteins described herein. Any of the polynucleotides describedherein may also comprise sequences (donor or patch sequences) fortargeted insertion into the HLA genes.

In yet another aspect, a gene delivery vector comprising any of thepolynucleotides described herein is provided. In certain embodiments,the vector is an adenovirus vector (e.g., an Ad5/F35 vector), alentiviral vector (LV) including integration competent orintegration-defective lentiviral vectors, or an adenovirus associatedviral vector (AAV). Thus, also provided herein are adenovirus (Ad)vectors, LV or adenovirus associate viral vectors (AAV) comprising asequence encoding at least one nuclease as described herein (e.g., ZFNor TALEN) and/or a donor sequence for targeted integration into a targetgene. In certain embodiments, the Ad vector is a chimeric Ad vector, forexample an Ad5/F35 vector. In certain embodiments, the lentiviral vectoris an integrase-defective lentiviral vector (IDLV) or an integrationcompetent lentiviral vector. In certain embodiments the vector ispseudo-typed with a VSV-G envelope, or with other envelopes.

In additional embodiments, the target gene is a gene (e.g., in a humancell) that regulates HLA expression (an HLA regulator gene). In certainembodiments, a CTIIA, a RFX5 gene, aTAP1, TAP2 or tapasin gene, orcombination thereof are targeted for regulation (e.g., activation,repression and/or inactivation). In some embodiments, the target geneencodes a microRNA capable of regulating HLA genes. The vectorsdescribed herein may also comprise donor sequences. In additionalembodiments, the donor sequences comprise human HLA genes or HLAregulator genes that are not endogenous to the host cell. In someembodiments, the HLA genes or HLA regulator genes of interest areinserted into the location of the endogenous HLA genes or HLA regulatorgenes, and in other embodiments the HLA genes or HLA regulator genes ofinterest are inserted into randomly selected loci, or into a separatelocus after genome-wide delivery. In some embodiments, the separatelocus for HLA transgene or HLA regulator transgene insertion is thePPP1R12C locus (see U.S Patent Publication Number 20080299580). In otherembodiments, the HLA transgene or HLA regulator transgene is insertedinto a CCR-5 locus. In some aspects, the donor comprises another nucleicacid of interest. By way of example only, this donor may contain a geneencoding a polypeptide of interest, or it may comprise a sequenceencoding a structural RNA (shRNA, miRNA, RNAi etc.). In someembodiments, cells are provided wherein an HLA gene or HLA regulatorgene of interest has been manipulated in a desired fashion (e.g.knocked-out, corrected etc.) and a donor and one or more additional ofZFNs and/or TALENs are provided to insert the donor into another locus(e.g. AAVS1).

In certain embodiments, a single vector comprises sequences encoding oneor more nucleases as described herein (e.g., ZFNs and/or TALENs) and thedonor sequence(s). In other embodiments, the donor sequence(s) arecontained in a first vector and the nuclease-encoding sequences arepresent in a second vector.

In yet another aspect, the disclosure provides a cell (e.g., an isolatedcell) comprising any of the proteins, polynucleotides and/or vectorsdescribed herein. In certain embodiments, the cell is selected from thegroup consisting of a stem/progenitor cell, a lymphocyte, a B cell, or aT-cell (e.g., CD4+ T-cell). In other embodiments, the cell is a cellfragment, including, but not limited to a platelet.

In another aspect, described herein are methods of inactivating an HLAgene or HLA regulator gene in a cell by introducing one or moreproteins, polynucleotides and/or vectors into the cell as describedherein. In any of these methods the nucleases may induce targetedmutagenesis, targeted deletions, targeted insertions of cellular DNAsequences, and/or facilitate targeted recombination at a predeterminedchromosomal locus. Thus, in certain embodiments, the nucleases (e.g.,ZFNs and/or TALENs) delete and/or insert one or more nucleotides at thetarget gene. In some embodiments the HLA gene is inactivated by nuclease(ZFN and/or TALEN) cleavage followed by non-homologous end joining. Inother embodiments, a genomic sequence in the target gene is replaced,for example using one or more pairs of ZFNs (or vector encoding saidZFNs) and/or one or more TALENs as described herein and a “donor”sequence that is inserted into the gene following targeted cleavage withthe ZFN(s) and/or TALEN(s). The donor sequence may be present in thenuclease fusion vector, present in a separate vector (e.g., Ad, AAV orLV vector) or, alternatively, may be introduced into the cell using adifferent nucleic acid delivery mechanism. In some embodiments, the ZFNsand/or TALENs are delivered using the mRNAs that encode them. In someembodiments, the nucleic acids may be delivered by electroporation oranother technique suitable for the delivery of naked nucleic acid.

In another aspect, methods of using the DNA-binding proteins and fusionsthereof for mutating an HLA gene and/or inactivating HLA function in acell or cell line are provided. Thus, a method for inactivating an HLAgene in a human cell is provided, the method comprising administering tothe cell any of the proteins or polynucleotides described herein.Methods are also provided herein for altering MHC function in any modelorganism.

In another aspect, the compositions and methods described herein can beused, for example, in the treatment or prevention or amelioration of anyHLA-related disorder (i.e., related to HLA haplotype). The methodstypically comprise (a) cleaving an endogenous HLA gene or HLA regulatorgene in an isolated cell (e.g., T-cell or lymphocyte) using a nuclease(e.g., ZFN or TALEN) such that the HLA or HLA regulator gene isinactivated; and (b) introducing the cell into the subject, therebytreating or preventing an HLA-related disorder. In certain embodiments,the HLA-related disorder is graft-versus-host disease (GVHD). Thenuclease(s) can be introduced as mRNA, in protein form and/or as a DNAsequence encoding the nuclease(s). In certain embodiments, the isolatedcell introduced into the subject further comprises additional genomicmodification, for example, an integrated exogenous sequence (into thecleaved HLA or HLA regulatory gene or a different gene, for example asafe harbor gene) and/or inactivation (e.g., nuclease-mediated) ofadditional genes, for example one or more TCR genes. The exogenoussequence may be introduced via a vector (e.g. Ad, AAV, LV), or by usinga technique such as electroporation. In some aspects, the compositionmay comprise isolated cell fragments and/or differentiated cells.

In some embodiments, nuclease fusions as described herein may beutilized for targeting stem cells such as induced pluripotent stem cells(iPSC), human embryonic stem cells (hES), mesenchymal stem cells (MSC),hematopoietic stem cells (HSC) or neuronal stem cells wherein theactivity of the nuclease fusion will result in an HLA allele containinga deletion. In some embodiments, the methods may be used to create stemcells in which more than one HLA gene has been altered. In otherembodiments, the invention provides methods for producing stem cellsthat have an HLA null phenotype. In some aspects, the stem cells may benull for one or more or all HLA class II gene expression. In otheraspects, the stem cells may be null for one or more or all HLA class Igene expression. In some aspects, the stem cells are null for all HLAgene expression. In other embodiments, the stem cells that have beenmodified at the HLA locus/loci are then differentiated.

Also provided are pharmaceutical compositions comprising the modifiedstem cells. Such pharmaceutical compositions may be usedprophylactically or therapeutically and may comprise iPSCs, hES, MSCs,HSCs or combinations and/or derivatives thereof. In other embodiments,cells, cell fragments (e.g., platelets) or tissues derived from suchmodified stem cells are provided such that such tissues are modified inthe HLA loci as desired. In some aspects, such cells are partiallydifferentiated (e.g. hematopoietic stem cells) while in others fullydifferentiated cells are provided (e.g. lymphocytes or megakarocytes)while in still others, fragments of differentiated cells are provided.In other embodiments, stem cells, and/or their differentiated progenyare provided that contain an altered HLA or HLA regulator gene or genes,and they also can contain an additional genetic modification including adeletion, alteration or insertion of a donor DNA at another locus ofinterest.

In some embodiments, cells treated with the DNA-binding domains orfusion proteins as described herein (e.g., ZFP-TF, TALE DNA bindingdomains TFs, ZFNs, and/or TALENs) may be mature cells such as CD4+ Tcells or NK cells. Such cells may comprise a protein comprising aDNA-binding domain as described herein for regulation of an HLA gene orHLA regulator gene, or may comprise a nuclease fusion (e.g., ZFN orTALEN) for introduction of a deletion and/or insertion into an HLA gene.In some aspects, such ZFN or TALEN comprising cells may additionallycomprise an exogenous DNA sequence. In some aspects, the mature cellsmay be used for cell therapy, for example, for a T cell transplant. Inother embodiments, the cells for use in T cell transplant containanother gene modification of interest. In one aspect, the T cellscontain an inserted chimeric antigen receptor (CAR) specific for acancer marker. In a further aspect, the inserted CAR is specific for theCD19 marker characteristic of B cell malignancies. Such cells would beuseful in a therapeutic composition for treating patients without havingto match HLA haplotype, and so would be able to be used as an“off-the-shelf” therapeutic for any patient in need thereof. In someaspects, cells in which genes encoding the TCRα and/or TCRβ chains havebeen manipulated or in which genes encoding TCR chains with desiredspecificity and affinity have been introduced are provided. In otherembodiments, HLA modified platelets are provided for therapeutic use intreatment of disorders such as thromobytopenia or other bleedingdisorders.

In still further aspects, the invention provides methods andcompositions for the generation of specific model systems for the studyof HLA disorders. In certain embodiments, models in which mutant HLAalleles are generated in embryonic stem cells for the generation of celland animal lines are provided. In certain embodiments, the model systemscomprise in vitro cell lines, while in other embodiments, the modelsystems comprise transgenic animals. In other embodiments, the inventionprovides methods and compositions for correcting a mutated HLA gene orHLA regulator and also provides methods and compositions for replacingone HLA allele with another.

In some embodiments, model systems are provided for HLA disorderswherein the target alleles (e.g., specific HLA haplotypes) are taggedwith expression markers. In certain embodiments, mutant alleles (e.g.,mutant HLA or HLA regulators) are tagged. In certain embodiments, themodel systems comprise in vitro cell lines, while in other embodiments,the model systems comprise transgenic animals.

Additionally, pharmaceutical compositions containing the nucleic acidsand/or DNA-binding domains (or fusion proteins comprising theDNA-binding domains) are also provided. For example, certaincompositions include a nucleic acid comprising a sequence that encodesone of the ZFPs and/or TALE DNA binding domains described hereinoperably linked to a regulatory sequence, combined with apharmaceutically acceptable carrier or diluent, wherein the regulatorysequence allows for expression or repression of the nucleic acid in acell. In certain embodiments, the ZFPs and/or TALE DNA binding domainsencoded are specific for an HLA allele. Protein based compositionsinclude one of more ZFPs TALE DNA binding domains as disclosed hereinand a pharmaceutically acceptable carrier or diluent.

Any of the methods described herein can be practiced in vitro, in vivoand/or ex vivo. In certain embodiments, the methods are practiced exvivo, for example to modify T-cells or NK cells prior to use fortreating a subject in need thereof.

These and other aspects will be readily apparent to the skilled artisanin light of disclosure as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic of some of the genes of the MEW Class I andClass II clusters.

FIGS. 2A and 2B depict gels displaying the results of Cel-I mismatchassays (Surveyor™, Transgenomics) for ZFNs targeting HLA A3 (FIG. 2A)and HLA A2 (FIG. 2B). The assay analyzes the percent mismatch with anamplified genomic DNA fragment, and thus allows quantitation of theamount of alterations (%) in the DNA sequence that occurred after NHEJrepair of the ZFN generated DSB. Both gels display the results for amock transfection, transfection with a GFP encoding plasmid, andtransfection with plasmids encoding the ZFN pairs indicated beneath thelanes of each gel into HEK293 cells. The percent gene modification isindicated at the bottom of the lanes.

FIGS. 3A through 3D depict the results of a FACS analysis of HEK293cells in which either HLA A2, HLA A3 or both HLA A2 and A3 have beendisrupted following specific ZFN treatment. FIG. 3A (“parental”) depictsthe parental HEK293 cells lacking any ZFN treatment. The column on theleft indicates staining for the HLA A2 allele while the column on theright indicates staining for the HLA A3 allele. The furthermost leftpeak in each panel indicates the isotype control (background). In theparental cells, the peak to the furthest right indicates the results forthe antibodies staining a B-LCL clone (positive control). The second tomost left peak indicates the amount of A2 or A3 positive cells prior tocytokine (IFN gamma) treatment, while the second furthest to the rightpeak indicates the results for HLA A2 or A3 expression followingcytokine treatment. FIGS. 3B to 3D, corresponding to cell clones 18-1(A2+A3-) (FIG. 3B), 8.18 (A2-A3+) (FIG. 3C) and 83 (A2-A3-) (FIG. 3D)contain the same analyses as shown in FIG. 3A (except the positive Bcell staining the line farthest to the right in the parental panels isnot shown). Thus, these data indicate that when the allele of interestis disrupted, no expression of the cell marker is observed, even uponcytokine stimulation.

FIGS. 4A and 4B are graphs depicting the analysis of cell lysis of theHLA-A knock-out HEK293 clones, 18-1, 8.18 and B cell clones (describedabove for FIG. 3) by specific CTLs targeting HLA A2 or HLA A3,respectively. In this experiment, CTLs restricted to either HLA A2 orHLA A3 were used in combination with the specific peptide epitope (SEQID NO:135 and SEQ ID NO:136) that, when displayed on the HLA complex,stimulates CTL action. FIG. 4A depicts the results when using HLA A3specific CTLs and HEK293 target cells with increasing concentrations oftheir peptide antigen. Cells that contain the unmodified A3 HLA geneproduct are lysed (parental, 8.18 and B-LCL). In FIG. 4B, a lysisexperiment was carried out using CTLs specific for HLA A2 in conjunctionwith a specific peptide antigen. When increasing concentrations ofpeptide are used, the CTLs are able to lyse those cells containing anintact HLA A2 gene product (parental HEK293, 18-1 and B-LCL). Thus, whenexpression of the HLA A gene targeted by the CTLs is disrupted, thecells are no longer susceptible to CTL directed lysis.

FIGS. 5A and 5B depict the results of a FACS analysis of HLA A2staining. In this experiment, primary T-cells were nucleofected withvarying amounts of mRNAs (2.5 μg shown in left panels, 5.0 μg shown inmiddle panels and 10 μg shown in right panels) encoding the HLA A2specific ZFNs. FIG. 5A depicts the results using standard cell cultureconditions following transfection, while FIG. 5B depicts the resultsusing the “transient cold shock” methodology. Up to nearly 42% of thecells can display an HLA A2 disruption characteristic using the “coldshock” methodology.

FIG. 6 depicts a gel displaying the results of a Cel-I mismatch analysisfor the primary T cells analyzed in FIG. 5. The lanes depict the resultsusing ZFN pairs with wild type Fok I catalytic domain (“wt”) and ZFNpairs with an “ELD/KKR” heterodimeric domains (“mut”) at the indicatedZFN concentrations (2.5 μg shown in left panels, 5.0 μg shown in middlepanels and 10 μg shown in right panels). The percent gene modificationdetected by this assay (“Targeted disruption (%)”) is shown in thebottom of each lane and the results are consistent with the FACSanalysis.

FIGS. 7A and 7B depict gels displaying the results of a Cel-I mismatchanalysis for ZFNs specific for the HLA C (FIG. 7A) and HLA B (FIG. 7B)genes. The arrows indicate the band on the gel indicative of genemodification.

FIGS. 8A and 8B depict gels displaying the results of a Cel-I mismatchanalysis for ZNFs specific for a target sequence downstream of the HLA Cgene (“HLA C-down”) and a target sequence upstream of the HLA B gene(“HLA B-up”). FIG. 8A depicts the results for the HLA C-down specificZFN pair where ZFNs containing a wild type FokI catalytic domain (wt)and the ZFN pair containing an EL/KK heterodimeric FokI domain (mut) areshown. The arrow depicts the band indicating the mismatch. The percentgene modification detected is shown at the bottom of each lane (“%NHEJ”). FIG. 8B depicts the results for two HLA B-up ZFN pairs where thearrow point out the bands indicative of gene modification.

FIGS. 9A and 9B illustrate an experiment designed to create a largedeletion that includes both the HLA B and the HLA C locus. FIG. 9A is aschematic of the HLA gene complex in the area of HLA B and HLA C, andthe regions targeted by the HLA B-up and HLA C-down ZFNs are indicated.The location of the primers used for the PCR to visualize the deletionare also indicated. FIG. 9B depicts the results of the deletion specificPCR following cleavage with the HLA B-up and HLA C-down ZFNs in K562cells. The lanes on the left side of the gel are a dilution series of adeletion PCR product that we inserted into a plasmid in order toquantitate the signal from the PCR. The deletion PCR was performed onDNAs isolated 3 and 10 days following nucleofection, and the ZFNs usedcontained either the wild-type (“wt”) or mutated (“mut”) FokI catalyticdomain (as discussed above in FIG. 8). The results indicate that at day3, approximately 5% of the alleles contained the HLA B and HLA Cdeletion.

FIGS. 10A and 10B depict gels displaying the results of cleavage usingZFNs targeting HLA regulatory genes in HEK293 cells. FIG. 10A depictsthe results of a Cel I mismatch assay with ZFNs targeted the TAP1 gene,while FIG. 10B depicts the results when the ZFNs targeted the TAP2 gene.ZFNs used are indicated above the appropriate lanes. These resultsindicate that these ZFNs are active in cleaving target DNA.

FIG. 11 depicts a gel displaying the results of a Cel-I mismatch assayusing ZFNs (ZFN numbers are indicated above lanes 1 and 2) targeting theTapasin gene in HEK293 cells. The percent gene modification is indicatedat the bottom of each lane. These data reveal that this ZFN pair isactive against this target.

FIG. 12 depicts a gel displaying the results of a Cel-I mismatch assayusing ZFNs targeting either a target location upstream of the DBP2 gene(DBP2up) or downstream of the DRA gene (DRAdown) in K562 cells. Thetransfections used the indicated ZFNs that contained either wild typeFokI catalytic domains (“wt”) or the EL/KK hetereodimeric FokI catalyticdomain (“mut”). The percent gene modification as measured by this assayis indicated at the bottom of each lane. The “wt” lanes show duplicatetransfections performed on two different transfection dates.

FIG. 13 depicts a gel displaying the results of a deletion PCR similarto that performed in FIG. 9. As described for FIG. 9, the lanes on theleft side of the gel contain a dilution series of subcloned PCR productfor use in quantitation of the frequency of alleles containing thedeletion. The deletion PCR was performed on DNA isolated from cells atday 3 or day 10 after the transfection, and the ZFNs used containedeither wild type Fok I catalytic domains (“wt”) or the EL/KK heterodimerFokI catalytic domains (“mut”). The results indicate that approximately0.04% of the alleles contained the large deletion.

FIG. 14 depicts sequencing results obtained by sequencing the deletionPCR product shown in FIG. 13. Lines 1-4 (SEQ ID NO:137) are individualnucleic acids from the PCR, while line 5 (SEQ ID NO:138) displays thegenomic sequence surrounding the 15909 ZFN target site and sequencedownstream thereof. Line 6 (SEQ ID NO:139) depicts the genomic sequencesurrounding the 15873 target site and sequence upstream thereof. Line 7(SEQ ID NO:137) shows the consensus sequence from lines 1-4. Theseresults indicate that following cleavage with the DBP2 up and DRA downZFNs, a large deletion has been generated by rejoining of the ends suchthat the resultant DNAs contain the distal target site of each ZFN pair.

FIG. 15 depicts a gel displaying the results following a Cel-I mismatchassay as described above. ZFNs targeting either the HLA class IIregulator genes CIITA or RFX5 were used in K562 cells, and the percentgene modification is indicated at the bottom of the lanes. Control lanescontain the results of experiments done either with no added ZFNs(“mock”) or transfected with a GFP encoding plasmid (“gfp”) are alsoshown.

FIG. 16 depicts a gel displaying the results following a Cel-I mismatchassay as described above. The CIITA targeting ZFNs were used in eitherK562 cells (“K”) or RAJI cells (“R”). The percent gene modification asdetected by this assay is shown at the bottom of the lanes, and thearrows show the bands indicative of modification activity. These resultsdemonstrate that the CIITA targeting ZFNs can work in RAJI cells as wellas K562 cells. “n.c.” indicates the negative control done without anyZFN encoding DNA during the transfection.

FIGS. 17A through 17D depict the results of a FACS analysis done on Tcells that had been previously been made transgenic for a chimericantigen receptor that targets CD19 (CD19CAR). FIG. 17A shows results ofisotype cells and FIG. 17B shows results when no mRNA was used. CD19CARmodified T cells were nucleofected with mRNAs encoding ZFNs that targetthe HLA A2 gene (FIG. 17C) and HLA-A2 negative cells were enriched bynegative bead sorting with an HLA-A2 antibody (FIG. 17D). FACS assay wasperformed using a HLA A2 specific antibody. The results indicate thatthe HLA A2 knock out cells were be enriched such that the populationcontained 95.3% HLA A2 knock outs (FIG. 17D).

FIG. 18 is a graph depicting the results of a lysis assay with HLAA2-specific CD19CAR containing T cells which had either been treatedwith the HLA A2-specific ZFNs (A2neg CD19RCAR-T cells) and enrichedusing the HLA-A2 specific antibody, or had been mock transfected(A2posCD19RCAR-T cells). Cells were incubated with increasing amounts ofthe specific peptide epitope. As a positive control, A2pos B cells wereused. The results indicate that the cells lacking the HLA A2 geneproduct are resistant to HLA A2-specific CTL induced lysis.

FIGS. 19A through 19G depict the results from several FACS analyses.mRNAs encoding ZFNs specific for either TCRβ (“TRBC,” FIG. 19A to FIG.19D) or TCRα (“TRAC,” FIG. 19E to FIG. 19G) were used in a range from2.5 μg to 10 μg as indicated above the FACS. The assay is designed toscore CD3 on the cell surface, a complex that is dependent on thepresence of the TCR. The results demonstrate that the TCRβ specific ZFNscan cause approximately 9% of the cells in the population to lose theCD3 marker while the TCRα specific ZFNs can cause approximately 28% ofcells to lose the CD3 marker.

FIGS. 20A and 20B depict a gel displaying the results of a Cel-I assayas described above to assess the amount of gene modification presentwhen either mRNAs encoding the TCRβ-specific ZFNs (TRBC, FIG. 20A) orencoding the TCRα-specific ZFNs (TRAC, FIG. 20B) are used. In thisexample, mRNAs were nucleofected and cultured according to eitherstandard conditions or using the “transient cold shock” conditions. Theresults agree generally with the results from FIG. 19 and indicate thatboth ZFN sets are capable of cleaving their intended targets.

DETAILED DESCRIPTION

Disclosed herein are DNA binding domains (e.g., ZFP and/or TALE DNAbinding proteins) and fusion proteins comprising these DNA bindingdomains (e.g., ZFNs, TALENs, ZFP-TFs and TALE-TFs) for targeting an HLAgene or an HLA regulator. The proteins described herein can repress oractivate a specific HLA gene, and change its expression. Similarly,DNA-binding proteins as described herein can target a HLA regulator andthrough modulating its expression, can cause a change in HLA expression.Also disclosed and provided herein are compositions including ZFNsand/or TALENs and methods for altering an HLA gene. These includecompositions and methods using engineered DNA-binding domains, i.e.,non-naturally occurring proteins which bind to a predetermined nucleicacid target sequence. The DNA binding domains and fusion proteinscomprising these DNA-binding domains as described herein can actefficiently and specifically on a desired HLA gene or genes, and canresult in a deletion of the specific gene and/or the introduction of analternate gene of interest into the targeted locus. Cells targeted inthis manner can be used as therapeutics, for example, transplants, orcan be used to generate either in vitro or in vivo model systems tostudy HLA gene function. Such cells can also be used as drug screeningtools to isolate and characterize small molecules or other types oftherapeutics for compounds that will act upon HLA expression. Cells canalso be generated in which following knock out of the desired HLA genes,other HLA genes may be inserted to change the HLA gene products that areexpressed on the cell's surface. Additionally, other genes of interestmay be inserted into cells in which the HLA genes have been manipulated.

Thus, the methods and compositions described herein provide methods fortreatment of HLA related disorders, and these methods and compositionscan comprise zinc finger transcription factors capable of modulatingtarget genes as well as engineered zinc finger nucleases.

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition,2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987 and periodic updates; the series METHODS INENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE ANDFUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe,eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

Definitions

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of correspondingnaturally-occurring amino acids.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹or lower. “Affinity” refers to the strength of binding: increasedbinding affinity being correlated with a lower K_(d).

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc fingerproteins have DNA-binding, RNA-binding and protein-binding activity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

A “TALE DNA binding domain” or “TALE” is a polypeptide comprising one ormore TALE repeat domains/units. The repeat domains are involved inbinding of the TALE to its cognate target DNA sequence. A single “repeatunit” (also referred to as a “repeat”) is typically 33-35 amino acids inlength and exhibits at least some sequence homology with other TALErepeat sequences within a naturally occurring TALE protein.

Zinc finger binding domains can be “engineered” to bind to apredetermined nucleotide sequence, for example via engineering (alteringone or more amino acids) of the recognition helix region of a naturallyoccurring zinc finger protein. Similarly, TALEs can be “engineered” tobind to a predetermined nucleotide sequence, for example by engineeringof the amino acids involved in DNA binding (the repeat variablediresidue or RVD region). Therefore, engineered zinc finger proteins orTALE proteins are proteins that are non-naturally occurring.Non-limiting examples of methods for engineering zinc finger proteinsand TALEs are design and selection. A designed protein is a protein notoccurring in nature whose design/composition results principally fromrational criteria. Rational criteria for design include application ofsubstitution rules and computerized algorithms for processinginformation in a database storing information of existing ZFP or TALEdesigns and binding data. See, for example, U.S. Pat. Nos. 6,140,081;6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059; WO98/53060; WO 02/016536 and WO 03/016496.

A “selected” zinc finger protein or TALE is a protein not found innature whose production results primarily from an empirical process suchas phage display, interaction trap or hybrid selection. See e.g., U.S.Pat. No. 5,789,538; U.S. Pat. No. 5,925,523; U.S. Pat. No. 6,007,988;U.S. Pat. No. 6,013,453; U.S. Pat. No. 6,200,759; WO 95/19431; WO96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970 WO 01/88197and WO 02/099084.

“Recombination” refers to a process of exchange of genetic informationbetween two polynucleotides. For the purposes of this disclosure,“homologous recombination (HR)” refers to the specialized form of suchexchange that takes place, for example, during repair of double-strandbreaks in cells via homology-directed repair mechanisms. This processrequires nucleotide sequence homology, uses a “donor” molecule totemplate repair of a “target” molecule (i.e., the one that experiencedthe double-strand break), and is variously known as “non-crossover geneconversion” or “short tract gene conversion,” because it leads to thetransfer of genetic information from the donor to the target. Withoutwishing to be bound by any particular theory, such transfer can involvemismatch correction of heteroduplex DNA that forms between the brokentarget and the donor, and/or “synthesis-dependent strand annealing,” inwhich the donor is used to resynthesize genetic information that willbecome part of the target, and/or related processes. Such specialized HRoften results in an alteration of the sequence of the target moleculesuch that part or all of the sequence of the donor polynucleotide isincorporated into the target polynucleotide.

In the methods of the disclosure, one or more targeted nucleases asdescribed herein create a double-stranded break in the target sequence(e.g., cellular chromatin) at a predetermined site, and a “donor”polynucleotide, having homology to the nucleotide sequence in the regionof the break, can be introduced into the cell. The presence of thedouble-stranded break has been shown to facilitate integration of thedonor sequence. The donor sequence may be physically integrated or,alternatively, the donor polynucleotide is used as a template for repairof the break via homologous recombination, resulting in the introductionof all or part of the nucleotide sequence as in the donor into thecellular chromatin. Thus, a first sequence in cellular chromatin can bealtered and, in certain embodiments, can be converted into a sequencepresent in a donor polynucleotide. Thus, the use of the terms “replace”or “replacement” can be understood to represent replacement of onenucleotide sequence by another, (i.e., replacement of a sequence in theinformational sense), and does not necessarily require physical orchemical replacement of one polynucleotide by another.

In any of the methods described herein, additional pairs of zinc-fingerproteins can be used for additional double-stranded cleavage ofadditional target sites within the cell.

In certain embodiments of methods for targeted recombination and/orreplacement and/or alteration of a sequence in a region of interest incellular chromatin, a chromosomal sequence is altered by homologousrecombination with an exogenous “donor” nucleotide sequence. Suchhomologous recombination is stimulated by the presence of adouble-stranded break in cellular chromatin, if sequences homologous tothe region of the break are present.

In any of the methods described herein, the first nucleotide sequence(the “donor sequence”) can contain sequences that are homologous, butnot identical, to genomic sequences in the region of interest, therebystimulating homologous recombination to insert a non-identical sequencein the region of interest. Thus, in certain embodiments, portions of thedonor sequence that are homologous to sequences in the region ofinterest exhibit between about 80 to 99% (or any integer therebetween)sequence identity to the genomic sequence that is replaced. In otherembodiments, the homology between the donor and genomic sequence ishigher than 99%, for example if only 1 nucleotide differs as betweendonor and genomic sequences of over 100 contiguous base pairs. Incertain cases, a non-homologous portion of the donor sequence cancontain sequences not present in the region of interest, such that newsequences are introduced into the region of interest. In theseinstances, the non-homologous sequence is generally flanked by sequencesof 50-1,000 base pairs (or any integral value therebetween) or anynumber of base pairs greater than 1,000, that are homologous oridentical to sequences in the region of interest. In other embodiments,the donor sequence is non-homologous to the first sequence, and isinserted into the genome by non-homologous recombination mechanisms.

Any of the methods described herein can be used for partial or completeinactivation of one or more target sequences in a cell by targetedintegration of donor sequence that disrupts expression of the gene(s) ofinterest. Cell lines with partially or completely inactivated genes arealso provided.

Furthermore, the methods of targeted integration as described herein canalso be used to integrate one or more exogenous sequences. The exogenousnucleic acid sequence can comprise, for example, one or more genes orcDNA molecules, or any type of coding or noncoding sequence, as well asone or more control elements (e.g., promoters). In addition, theexogenous nucleic acid sequence may produce one or more RNA molecules(e.g., small hairpin RNAs (shRNAs), inhibitory RNAs (RNAis), microRNAs(miRNAs), etc.).

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

A “cleavage half-domain” is a polypeptide sequence which, in conjunctionwith a second polypeptide (either identical or different) forms acomplex having cleavage activity (preferably double-strand cleavageactivity). The terms “first and second cleavage half-domains;” “+ and−cleavage half-domains” and “right and left cleavage half-domains” areused interchangeably to refer to pairs of cleavage half-domains thatdimerize.

An “engineered cleavage half-domain” is a cleavage half-domain that hasbeen modified so as to form obligate heterodimers with another cleavagehalf-domain (e.g., another engineered cleavage half-domain). See, also,U.S. Patent Publication Nos. 2005/0064474, 20070218528 and 2008/0131962,incorporated herein by reference in their entireties.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 10,000 nucleotides inlength (or any integer value therebetween or thereabove), preferablybetween about 100 and 1,000 nucleotides in length (or any integertherebetween), more preferably between about 200 and 500 nucleotides inlength.

“Chromatin” is the nucleoprotein structure comprising the cellulargenome. Cellular chromatin comprises nucleic acid, primarily DNA, andprotein, including histones and non-histone chromosomal proteins. Themajority of eukaryotic cellular chromatin exists in the form ofnucleosomes, wherein a nucleosome core comprises approximately 150 basepairs of DNA associated with an octamer comprising two each of histonesH2A, H2B, H3 and H4; and linker DNA (of variable length depending on theorganism) extends between nucleosome cores. A molecule of histone H1 isgenerally associated with the linker DNA. For the purposes of thepresent disclosure, the term “chromatin” is meant to encompass all typesof cellular nucleoprotein, both prokaryotic and eukaryotic. Cellularchromatin includes both chromosomal and episomal chromatin.

A “chromosome,” is a chromatin complex comprising all or a portion ofthe genome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist. For example, thesequence 5′ GAATTC 3′ is a target site for the Eco RI restrictionendonuclease. Exemplary target sites for various targeted ZFPs are shownin Table 2.

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer. Anexogenous molecule can also be the same type of molecule as anendogenous molecule but derived from a different species than the cellis derived from. For example, a human nucleic acid sequence may beintroduced into a cell line originally derived from a mouse or hamster.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPDNA-binding domain and one or more activation domains) and fusionnucleic acids (for example, a nucleic acid encoding the fusion proteindescribed supra). Examples of the second type of fusion moleculeinclude, but are not limited to, a fusion between a triplex-formingnucleic acid and a polypeptide, and a fusion between a minor groovebinder and a nucleic acid.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of an mRNA. Gene products also include RNAswhich are modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

“Modulation” of gene expression refers to a change in the activity of agene. Modulation of expression can include, but is not limited to, geneactivation and gene repression. Genome editing (e.g., cleavage,alteration, inactivation, random mutation) can be used to modulateexpression. Gene inactivation refers to any reduction in gene expressionas compared to a cell that does not include a ZFP as described herein.Thus, gene inactivation may be partial or complete.

A “region of interest” is any region of cellular chromatin, such as, forexample, a gene or a non-coding sequence within or adjacent to a gene,in which it is desirable to bind an exogenous molecule. Binding can befor the purposes of targeted DNA cleavage and/or targeted recombination.A region of interest can be present in a chromosome, an episome, anorganellar genome (e.g., mitochondrial, chloroplast), or an infectingviral genome, for example. A region of interest can be within the codingregion of a gene, within transcribed non-coding regions such as, forexample, leader sequences, trailer sequences or introns, or withinnon-transcribed regions, either upstream or downstream of the codingregion. A region of interest can be as small as a single nucleotide pairor up to 2,000 nucleotide pairs in length, or any integral value ofnucleotide pairs.

“Eukaryotic” cells include, but are not limited to, fungal cells (suchas yeast), plant cells, animal cells, mammalian cells and human cells(e.g., T-cells).

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a ZFPDNA-binding domain is fused to an activation domain, the ZFP DNA-bindingdomain and the activation domain are in operative linkage if, in thefusion polypeptide, the ZFP DNA-binding domain portion is able to bindits target site and/or its binding site, while the activation domain isable to up-regulate gene expression. When a fusion polypeptide in whicha ZFP DNA-binding domain is fused to a cleavage domain, the ZFPDNA-binding domain and the cleavage domain are in operative linkage if,in the fusion polypeptide, the ZFP DNA-binding domain portion is able tobind its target site and/or its binding site, while the cleavage domainis able to cleave DNA in the vicinity of the target site.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain oneore more amino acid or nucleotide substitutions. Methods for determiningthe function of a nucleic acid (e.g., coding function, ability tohybridize to another nucleic acid) are well-known in the art. Similarly,methods for determining protein function are well-known. For example,the DNA-binding function of a polypeptide can be determined, forexample, by filter-binding, electrophoretic mobility-shift, orimmunoprecipitation assays. DNA cleavage can be assayed by gelelectrophoresis. See Ausubel et al., supra. The ability of a protein tointeract with another protein can be determined, for example, byco-immunoprecipitation, two-hybrid assays or complementation, bothgenetic and biochemical. See, for example, Fields et al. (1989) Nature340:245-246; U.S. Pat. No. 5,585,245 and PCT WO 98/44350.

A “vector” is capable of transferring gene sequences to target cells.Typically, “vector construct,” “expression vector,” and “gene transfervector,” mean any nucleic acid construct capable of directing theexpression of a gene of interest and which can transfer gene sequencesto target cells. Thus, the term includes cloning, and expressionvehicles, as well as integrating vectors.

A “reporter gene” or “reporter sequence” refers to any sequence thatproduces a protein product that is easily measured, preferably althoughnot necessarily in a routine assay. Suitable reporter genes include, butare not limited to, sequences encoding proteins that mediate antibioticresistance (e.g., ampicillin resistance, neomycin resistance, G418resistance, puromycin resistance), sequences encoding colored orfluorescent or luminescent proteins (e.g., green fluorescent protein,enhanced green fluorescent protein, red fluorescent protein,luciferase), and proteins which mediate enhanced cell growth and/or geneamplification (e.g., dihydrofolate reductase). Epitope tags include, forexample, one or more copies of FLAG, His, myc, Tap, HA or any detectableamino acid sequence. “Expression tags” include sequences that encodereporters that may be operably linked to a desired gene sequence inorder to monitor expression of the gene of interest.

DNA-Binding Domains

Described herein are compositions comprising a DNA-binding domain thatspecifically bind to a target site in any gene comprising a HLA gene ora HLA regulator. Any DNA-binding domain can be used in the compositionsand methods disclosed herein.

In certain embodiments, the DNA binding domain comprises a zinc fingerprotein. Preferably, the zinc finger protein is non-naturally occurringin that it is engineered to bind to a target site of choice. See, forexample, Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al.(2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) NatureBiotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol.12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416;U.S. Pat. Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558;7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635;7,253,273; and U.S. Patent Publication Nos. 2005/0064474; 2007/0218528;2005/0267061, all incorporated herein by reference in their entireties.

An engineered zinc finger binding domain can have a novel bindingspecificity, compared to a naturally-occurring zinc finger protein.Engineering methods include, but are not limited to, rational design andvarious types of selection. Rational design includes, for example, usingdatabases comprising triplet (or quadruplet) nucleotide sequences andindividual zinc finger amino acid sequences, in which each triplet orquadruplet nucleotide sequence is associated with one or more amino acidsequences of zinc fingers which bind the particular triplet orquadruplet sequence. See, for example, co-owned U.S. Pat. Nos. 6,453,242and 6,534,261, incorporated by reference herein in their entireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB2,338,237. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-owned WO02/077227.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-owned WO02/077227.

Selection of target sites; ZFPs and methods for design and constructionof fusion proteins (and polynucleotides encoding same) are known tothose of skill in the art and described in detail in U.S. Pat. Nos.6,140,081; 5,789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988;6,013,453; 6,200,759; WO 95/19431; WO 96/06166; WO 98/53057; WO98/54311; WO 00/27878; WO 01/60970 WO 01/88197; WO 02/099084; WO98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein.

Alternatively, the DNA-binding domain may be derived from a nuclease.For example, the recognition sequences of homing endonucleases andmeganucleases such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI,I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIIIare known. See also U.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252;Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al.(1989) Gene 82:115-118; Perler et al. (1994) Nucleic Acids Res. 22,1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al. (1996)J. Mol. Biol. 263:163-180; Argast et al. (1998) J. Mol. Biol.280:345-353 and the New England Biolabs catalogue. In addition, theDNA-binding specificity of homing endonucleases and meganucleases can beengineered to bind non-natural target sites. See, for example, Chevalieret al. (2002) Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic AcidsRes. 31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques etal. (2007) Current Gene Therapy 7:49-66; U.S. Patent Publication No.20070117128.

In certain embodiments, the DNA binding domain is an engineered zincfinger protein that binds (in a sequence-specific manner) to a targetsite in a HLA gene or HLA regulatory gene and modulates expression ofHLA. The ZFPs can bind selectively to a specific haplotype of interest.For a discussion of HLA haplotypes identified in the United Statespopulation and their frequency according to different races, see Maierset al (2007) Human Immunology 68: 779-788, incorporated by referenceherein. Additionally, ZFPs are provided that bind to functional HLAregulator genes including, but not limited to, Tap1, Tap2, Tapascin,CTFIIA, and RFX5. HLA target sites typically include at least one zincfinger but can include a plurality of zinc fingers (e.g., 2, 3, 4, 5, 6or more fingers). Usually, the ZFPs include at least three fingers.Certain of the ZFPs include four, five or six fingers. The ZFPs thatinclude three fingers typically recognize a target site that includes 9or 10 nucleotides; ZFPs that include four fingers typically recognize atarget site that includes 12 to 14 nucleotides; while ZFPs having sixfingers can recognize target sites that include 18 to 21 nucleotides.The ZFPs can also be fusion proteins that include one or more regulatorydomains, which domains can be transcriptional activation or repressiondomains.

Specific examples of targeted ZFPs are disclosed in Table 1. The firstcolumn in this table is an internal reference name (number) for a ZFPand corresponds to the same name in column 1 of Table 2. “F” refers tothe finger and the number following “F” refers which zinc finger (e.g.,“F1” refers to finger 1).

TABLE 1 Zinc finger proteins Target SBS # F1 F2 F3 F4 F5 F6 Class IHLA A2 18889 QSSHLTR RSDHLTT RSDTLSQ RSADLSR QSSDLSR RSDALTQ (SEQ ID(SEQ (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 1) NO: 2) NO: 3) NO: 4) NO: 5)NO: 6) HLA A2 18881 QKTHLAK RSDTLSN RKDVRIT RSDHLST DSSARKK NA (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 7) NO: 8) NO: 9) NO: 10) NO: 11)HLA A2 24859 QNAHRKT RSDSLLR RNDDRKK RSDHLST DSSARKK NA (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: 12) NO: 13) NO: 14) NO: 10) NO: 11) HLA A325191 DRSHLSR RSDDLTR DRSDLSR QSGHLSR NA NA (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 15) NO: 16) NO: 17) NO: 18) HLA A3 25190 DRSALSR QSSDLRRDRSALSR DRSHLAR RSDDLSK DRSHLAR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 19) NO: 20) NO: 19) NO: 21) NO: 22) NO: 21) HLA B 25316SSELLNE TSSHLSR QSGDRNK RSANLAR RSDNLRE NA (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 23) NO: 24) NO: 25) NO: 26) NO: 27) HLA B 25317QSGDLTR RSDDLTR DQSTLRN DRSNLSR DAFTRTR NA (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 28) NO: 16) NO: 29) NO: 30) NO: 31) HLA B-up 15267RSDNLSE ASKTRKN TSGNLTR RSDALAR NA NA (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: 32) NO: 33) NO: 34) NO: 35) HLA B-up 15265 DRSALSR QSGNLAR DRSALSRQSGHLSR NA NA (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 19) NO: 36) NO: 19)NO: 18) HLA B-up 17454 RSDNLSE ASKTRKN QSGHLSR TSGHLSR QSGHLSR NA(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 32) NO: 33) NO: 18) NO: 37)NO: 18) HLA B-up 17456 RSADLTR QSGDLTR QSGNLAR QSGDLTR NA NA (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: 38) NO: 28) NO: 36) NO: 28) HLA C-down 15296QSGHLSR RSDHLST QSADRTK TSGSLSR QSADRTK NA (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 18) NO: 10) NO: 39) NO: 40) NO: 39) HLA C-down 15298QSGDLTR RSDHLST QSADRTK RSDNLSA RSDNRTT NA (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 28) NO: 10) NO: 39) NO: 41) NO: 42) HLA C 25588QRSNLVR DRSALAR QSSDLRR RSDDLTR RSDDLTR NA (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 43) NO: 44) NO: 20) NO: 16) NO: 16) HLA C 25589RSDDLTR DRSDLSR QSGHLSR RSDHLSA ESRYLMV NA (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 16) NO: 17) NO: 18) NO: 45) NO: 46) Class II DBP2-up15872 RSDHLST DNANRTK QSGDLTR RSDALST ASSNRKT NA (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 10) NO: 47) NO: 28) NO: 48) NO: 49) DBP2-up 15873TSGNLTR DRSDLSR RSDNLSE RSANLTR QSGHLSR NA (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 34) NO: 17) NO: 32) NO: 50) NO: 18) DRA-down 15909RSDNLSE TSGSLTR TSGHLSR RSDNLSQ ASNDRKK NA (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 32) NO: 51) NO: 37) NO: 52) NO: 53) DRA-down 15910 RSDNLSRDNNARIN RSDSLSV QNQHRIN RSDHLSR NA (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 54) NO: 55) NO: 56) NO: 57) NO: 58) Regulators TAP1 28386DSSDRKK DRSHLTR RSDALAR QSSDLSR RSDNLTT NA (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 59) NO: 60) NO: 35) NO: 5) NO: 61) TAP1 28385RSANLAR QSGHLSR TSGNLTR QSGALVI RSDHLSE RKHDRTK (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: 26) NO: 18) NO: 34) NO: 62) NO: 63) NO: 64)TAP2 28394 QSSDLSR QSGDLTR QSSHLTR RSDDRKT TSGNLTR RSDDLTR (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 5) NO: 28) NO: 1) NO: 65)NO: 34) NO: 16) TAP2 28393 RSDNLST RSDALAR RSDVLSA DRSNRIK RREDLITTSSNLSR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 66) NO: 35)NO: 67) NO: 68) NO: 69) NO: 70) Tapasin 28406 RSDNLSE KRCNLRC DRSDLSRQTTHRNR DRSDLSR QSSTRAR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: 32) NO: 71) NO: 17) NO: 72) NO: 17) NO: 73) Tapasin 28404 QSSDLSRRSDNLTR QSSHLTR QSSDLTR RSDNLAR QKVNLMS (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 5) NO: 74) NO: 1) NO: 75) NO: 76) NO: 77) Tapasin28403 TSGNLTR LSQDLNR RSDSLSA DRSHLAR RSDHLST QSGHLSR (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 34) NO: 78) NO: 79) NO: 21) NO: 10)NO: 18) CTIIA 15486 RSDDLTR SSSNLTK TSGSLSR QSGDLTR RSDHLSE RNRDRIT(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 16) NO: 80) NO: 40)NO: 28) NO: 63) NO: 81) CTIIA 15486 RSDDLTR RSDHLSE NSRNRKT RSDNLSQASNDRKK NA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 16) NO: 63)NO: 82) NO: 52) NO: 53) CTIIA 15487 RSDDLSR RNDDRKK DRSDLSR RSDHLSEARSTRTN NA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 83) NO: 14)NO: 17) NO: 63) NO: 84) RFX5 TSGNLTR QSGNLAR RSDHLTQ ASMALNE TSSNLSR NA(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 34) NO: 36) NO: 85) NO: 86)NO: 70) RFX5 15507 RSDVLSE RNQHRKT RSDHLST QSSDLRR RSDNLST RSADRKN(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 87) NO: 88) NO: 10)NO: 20) NO: 66) NO: 89) Others TRAC 25539 QSGDLTR QWGTRYR ERGTLARRSDNLRE QSGDLTR TSGSLTR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: 28) NO: 90) NO: 91) NO: 27) NO: 28) NO: 51) TRAC 25540 QSGDLTRWRSSLAS QSGDLTR HKWVLRQ DRSNLTR NA (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 28) NO: 92) NO: 28) NO: 93) NO: 94) TRBC 16783 RSDVLSADRSNRIK RSDVLSE QSGNLAR QSGSLTR NA (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 67) NO: 68) NO: 87) NO: 36) NO: 95) TRBC 16787 RSDHLSTRSDNLTR DRSNLSR TSSNRKT RSANLAR RNDDRKK (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 10) NO: 74) NO: 30) NO: 96) NO: 26) NO: 14)

The sequence for the target sites of these proteins are disclosed inTable 2. Table 2 shows target sequences for the indicated zinc fingerproteins. Nucleotides in the target site that are contacted by the ZFPrecognition helices are indicated in uppercase letters; non-contactednucleotides indicated in lowercase.

TABLE 2 Zinc finger target sites Target SBS # Target site Class I HLA A218889 gtATGGCTGCGACGTGGGGTcggacggg_(SEQ ID NO: 97) HLA A2 18881ttATCTGGATGGTGTGAgaacctggccc_(SEQ ID NO: 98) HLA A2 24859tcCTCTGGACGGTGTGAgaacctggccc_(SEQ ID NO: 99) HLA A3 25191atGGAGCCGCGGGCgccgtggatagagc_(SEQ ID NO: 100) HLA A3 25190ctGGCTCGcGGCGTCGCTGTCgaaccgc_(SEQ ID NO: 101) HLA B-up 25316tcCAGGAGcTCAGGTCCTcgttcagggc_(SEQ ID NO: 102) HLA B-up 25317cgGCGGACACCGCGGCTcagatcaccca_(SEQ ID NO: 103) HLA B-up 15267agGTGGATGCCCAGgacgagctttgagg_(SEQ ID NO: 104) HLA B-up 15265agGGAGCAGAAGCAgcgcagcagcgcca_(SEQ ID NO: 105) HLA B-up 17454ctGGAGGTGGAtGCCCAGgacgagcttt_(SEQ ID NO: 106) HLA B-up 17456gaGCAGAAGCAGCGcagcagcgccacct_(SEQ ID NO: 107) HLA C-down 15296ccTCAGTTTCATGGGGAttcaagggaac_(SEQ ID NO: 108) HLA C-down 15298ccTAGGAGgtcatgggcaTTTGCCATGC_(SEQ ID NO: 109) HLA C-down 25588tcGCGGCGtcGCTGTCGAAccgcacgaa_(SEQ ID NO: 110) HLA C-down 25589ccAAGAGGGGAGCCGCGggagccgtggg_(SEQ ID NO: 111) Class II DBP2-up 15872gaAATAAGGCATACTGGtattactaatg_(SEQ ID NO: 112) DBP2-up 15873gaGGAGAGCAGGCCGATtacctgaccca_(SEQ ID NO: 113) DRA-down 15909tcTCCCAGGGTgGTTCAGtggcagaatt_(SEQ ID NO: 114) DRA-down 15910gcGGGGGAAAGaGAGGAGgagagaagga_(SEQ ID NO: 115) Regulators TAP1 28386agAAGGCTGTGGGCTCCtcagagaaaat_(SEQ ID NO: 116) TAP1 28385acTCTGGGGTAGATGGAGAGcagtacct_(SEQ ID NO: 117) TAP2 28394ttGCGGATCCGGGAGCAGCTtttctcct_(SEQ ID NO: 118) TAP2 28393ttGATTCGaGACATGGTGTAGgtgaagc_(SEQ ID NO: 119) Tapasin 28406ccACAGCCAGAGCCtCAGCAGgagcctg_(SEQ ID NO: 120) Tapasin 28405cgCAAGAGGCTGGAGAGGCTgaggactg_(SEQ ID NO: 121) Tapasin 28404ctGGATGGGGCTTGGCTGATggtcagca_(SEQ ID NO: 122) Tapasin 28403gcCCGCGGGCAGTTcTGCGCGggggtca_(SEQ ID NO: 123) CTIIA 15486gcTCCCAGgCAGCGGGCGggaggctgga_(SEQ ID NO: 124) CTIIA 15487ctACTCGGGCCaTCGGCGgctgcctcgg_(SEQ ID NO: 125) RFX5 15506ttGATGTCAGGGAAGATctctctgatga_(SEQ ID NO: 126) RFX5 15507gcTCGAAGGCTTGGTGGCCGgggccagt_(SEQ ID NO: 127) Others TRAC 25539ttGTTGCTcCAGGCCACAGCActgttgc_(SEQ ID NO: 128) TRAC 25540ctGACTTTGCATGTGCAaacgccttcaa_(SEQ ID NO: 129) TRBC 16783ccGTAGAACTGGACTTGacagcggaagt_(SEQ ID NO: 130) TRBC 16787tcTCGGAGAATGACGAGTGGacccagga_(SEQ ID NO: 131)

In some embodiments, the DNA binding domain is an engineered domain froma TAL effector similar to those derived from the plant pathogensXanthomonas (see Boch et al, (2009) Science 326: 1509-1512 and Moscouand Bogdanove, (2009) Science 326: 1501) and Ralstonia (see Heuer et al(2007) Applied and Environmental Microbiology 73(13): 4379-4384); U.S.Pat. Nos. 8,586,526 and 8,586,363.

Fusion Proteins

Fusion proteins comprising DNA-binding proteins (e.g., ZFPs or TALEs) asdescribed herein and a heterologous regulatory (functional) domain (orfunctional fragment thereof) are also provided. Common domains include,e.g., transcription factor domains (activators, repressors,co-activators, co-repressors), silencers, oncogenes (e.g., myc, jun,fos, myb, max, mad, rel, ets, bcl, myb, mos family members etc.); DNArepair enzymes and their associated factors and modifiers; DNArearrangement enzymes and their associated factors and modifiers;chromatin associated proteins and their modifiers (e.g. kinases,acetylases and deacetylases); and DNA modifying enzymes (e.g.,methyltransferases, topoisomerases, helicases, ligases, kinases,phosphatases, polymerases, endonucleases) and their associated factorsand modifiers. U.S. Patent Publication Nos. 20050064474; 20060188987 and2007/0218528 for details regarding fusions of DNA-binding domains andnuclease cleavage domains, incorporated by reference in their entiretiesherein

Suitable domains for achieving activation include the HSV VP16activation domain (see, e.g., Hagmann et al., J. Virol. 71, 5952-5962(1997)) nuclear hormone receptors (see, e.g., Torchia et al., Curr.Opin. Cell. Biol. 10:373-383 (1998)); the p65 subunit of nuclear factorkappa B (Bitko & Barik, J. Virol. 72:5610-5618 (1998) and Doyle & Hunt,Neuroreport 8:2937-2942 (1997)); Liu et al., Cancer Gene Ther. 5:3-28(1998)), or artificial chimeric functional domains such as VP64 (Beerliet al., (1998) Proc. Natl. Acad. Sci. USA 95:14623-33), and degron(Molinari et al., (1999) EMBO J. 18, 6439-6447). Additional exemplaryactivation domains include, Oct 1, Oct-2A, Sp1, AP-2, and CTF1 (Seipelet al., EMBO J. 11, 4961-4968 (1992) as well as p300, CBP, PCAF, SRC1PvALF, AtHD2A and ERF-2. See, for example, Robyr et al. (2000) Mol.Endocrinol. 14:329-347; Collingwood et al. (1999) J. Mol. Endocrinol.23:255-275; Leo et al. (2000) Gene 245:1-11; Manteuffel-Cymborowska(1999) Acta Biochim. Pol. 46:77-89; McKenna et al. (1999) J. SteroidBiochem. Mol. Biol. 69:3-12; Malik et al. (2000) Trends Biochem. Sci.25:277-283; and Lemon et al. (1999) Curr. Opin. Genet. Dev. 9:499-504.Additional exemplary activation domains include, but are not limited to,OsGAI, HALF-1, C1, AP1, ARF-5,-6,-7, and -8, CPRF1, CPRF4, MYC-RP/GP,and TRAB1. See, for example, Ogawa et al. (2000) Gene 245:21-29; Okanamiet al. (1996) Genes Cells 1:87-99; Goff et al. (1991) Genes Dev.5:298-309; Cho et al. (1999) Plant Mol. Biol. 40:419-429; Ulmason et al.(1999) Proc. Natl. Acad. Sci. USA 96:5844-5849; Sprenger-Haussels et al.(2000) Plant J. 22:1-8; Gong et al. (1999) Plant Mol. Biol. 41:33-44;and Hobo et al. (1999) Proc. Natl. Acad. Sci. USA 96:15,348-15,353.

It will be clear to those of skill in the art that, in the formation ofa fusion protein (or a nucleic acid encoding same) between a DNA-bindingdomain and a functional domain, either an activation domain or amolecule that interacts with an activation domain is suitable as afunctional domain. Essentially any molecule capable of recruiting anactivating complex and/or activating activity (such as, for example,histone acetylation) to the target gene is useful as an activatingdomain of a fusion protein. Insulator domains, localization domains, andchromatin remodeling proteins such as ISWI-containing domains and/ormethyl binding domain proteins suitable for use as functional domains infusion molecules are described, for example, in co-owned U.S. PatentPublications 2002/0115215 and 2003/0082552 and in co-owned WO 02/44376.

Exemplary repression domains include, but are not limited to, KRAB A/B,KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3,members of the DNMT family (e.g., DNMT1, DNMT3A, DNMT3B), Rb, and MeCP2.See, for example, Bird et al. (1999) Cell 99:451-454; Tyler et al.(1999) Cell 99:443-446; Knoepfler et al. (1999) Cell 99:447-450; andRobertson et al. (2000) Nature Genet. 25:338-342. Additional exemplaryrepression domains include, but are not limited to, ROM2 and AtHD2A.See, for example, Chem et al. (1996) Plant Cell 8:305-321; and Wu et al.(2000) Plant J. 22:19-27.

Fusion molecules are constructed by methods of cloning and biochemicalconjugation that are well known to those of skill in the art. Fusionmolecules comprise a DNA-binding domain and a functional domain (e.g., atranscriptional activation or repression domain). Fusion molecules alsooptionally comprise nuclear localization signals (such as, for example,that from the SV40 medium T-antigen) and epitope tags (such as, forexample, FLAG and hemagglutinin). Fusion proteins (and nucleic acidsencoding them) are designed such that the translational reading frame ispreserved among the components of the fusion.

Fusions between a polypeptide component of a functional domain (or afunctional fragment thereof) on the one hand, and a non-proteinDNA-binding domain (e.g., antibiotic, intercalator, minor groove binder,nucleic acid) on the other, are constructed by methods of biochemicalconjugation known to those of skill in the art. See, for example, thePierce Chemical Company (Rockford, Ill.) Catalogue. Methods andcompositions for making fusions between a minor groove binder and apolypeptide have been described. Mapp et al. (2000) Proc. Natl. Acad.Sci. USA 97:3930-3935.

In certain embodiments, the target site bound by the zinc finger proteinis present in an accessible region of cellular chromatin. Accessibleregions can be determined as described, for example, in co-ownedInternational Publication WO 01/83732. If the target site is not presentin an accessible region of cellular chromatin, one or more accessibleregions can be generated as described in co-owned WO 01/83793. Inadditional embodiments, the DNA-binding domain of a fusion molecule iscapable of binding to cellular chromatin regardless of whether itstarget site is in an accessible region or not. For example, suchDNA-binding domains are capable of binding to linker DNA and/ornucleosomal DNA. Examples of this type of “pioneer” DNA binding domainare found in certain steroid receptor and in hepatocyte nuclear factor 3(HNF3). Cordingley et al. (1987) Cell 48:261-270; Pina et al. (1990)Cell 60:719-731; and Cirillo et al. (1998) EMBO J. 17:244-254.

The fusion molecule may be formulated with a pharmaceutically acceptablecarrier, as is known to those of skill in the art. See, for example,Remington's Pharmaceutical Sciences, 17th ed., 1985; and co-owned WO00/42219.

The functional component/domain of a fusion molecule can be selectedfrom any of a variety of different components capable of influencingtranscription of a gene once the fusion molecule binds to a targetsequence via its DNA binding domain. Hence, the functional component caninclude, but is not limited to, various transcription factor domains,such as activators, repressors, co-activators, co-repressors, andsilencers.

Additional exemplary functional domains are disclosed, for example, inco-owned U.S. Pat. No. 6,534,261 and US Patent Publication No.2002/0160940.

Functional domains that are regulated by exogenous small molecules orligands may also be selected. For example, RheoSwitch® technology may beemployed wherein a functional domain only assumes its activeconformation in the presence of the external RheoChem™ ligand (see forexample US 20090136465). Thus, the ZFP may be operably linked to theregulatable functional domain wherein the resultant activity of theZFP-TF is controlled by the external ligand.

Nucleases

In certain embodiments, the fusion protein comprises a DNA-bindingbinding domain and cleavage (nuclease) domain. As such, genemodification can be achieved using a nuclease, for example an engineerednuclease. Engineered nuclease technology is based on the engineering ofnaturally occurring DNA-binding proteins. For example, engineering ofhoming endonucleases with tailored DNA-binding specificities has beendescribed. Chames et al. (2005) Nucleic Acids Res 33(20):e178; Arnouldet al. (2006) J. Mol. Biol. 355:443-458. In addition, engineering ofZFPs has also been described. See, e.g., U.S. Pat. Nos. 6,534,261;6,607,882; 6,824,978; 6,979,539; 6,933,113; 7,163,824; and 7,013,219.

In addition, ZFPs and/or TALEs have been fused to nuclease domains tocreate ZFNs and TALENs—a functional entity that is able to recognize itsintended nucleic acid target through its engineered (ZFP or TALE) DNAbinding domain and cause the DNA to be cut near the DNA binding site viathe nuclease activity. See, e.g., Kim et al. (1996) Proc Nat'l Acad SciUSA 93(3):1156-1160. More recently, such nucleases have been used forgenome modification in a variety of organisms. See, for example, UnitedStates Patent Publications 20030232410; 20050208489; 20050026157;20050064474; 20060188987; 20060063231; and International Publication WO07/014275.

Thus, the methods and compositions described herein are broadlyapplicable and may involve any nuclease of interest. Non-limitingexamples of nucleases include meganucleases, TALENs and zinc fingernucleases. The nuclease may comprise heterologous DNA-binding andcleavage domains (e.g., zinc finger nucleases; meganuclease DNA-bindingdomains with heterologous cleavage domains) or, alternatively, theDNA-binding domain of a naturally-occurring nuclease may be altered tobind to a selected target site (e.g., a meganuclease that has beenengineered to bind to site different than the cognate binding site).

In certain embodiments, the nuclease is a meganuclease (homingendonuclease). Naturally-occurring meganucleases recognize 15-40base-pair cleavage sites and are commonly grouped into four families:the LAGLIDADG (SEQ ID NO:141) family, the GIY-YIG family, the His-Cystbox family and the HNH family. Exemplary homing endonucleases includeI-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII,I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII. Theirrecognition sequences are known. See also U.S. Pat. No. 5,420,032; U.S.Pat. No. 6,833,252; Belfort et al. (1997) Nucleic Acids Res.25:3379-3388; Dujon et al. (1989) Gene 82:115-118; Perler et al. (1994)Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet. 12:224-228;Gimble et al. (1996) J. Mol. Biol. 263:163-180; Argast et al. (1998) J.Mol. Biol. 280:345-353 and the New England Biolabs catalogue.

DNA-binding domains from naturally-occurring meganucleases, primarilyfrom the LAGLIDADG (SEQ ID NO:141) family, have been used to promotesite-specific genome modification in plants, yeast, Drosophila,mammalian cells and mice, but this approach has been limited to themodification of either homologous genes that conserve the meganucleaserecognition sequence (Monet et al. (1999), Biochem. Biophysics. Res.Common. 255: 88-93) or to pre-engineered genomes into which arecognition sequence has been introduced (Route et al. (1994), Mol.Cell. Biol. 14: 8096-106; Chilton et al. (2003), Plant Physiology. 133:956-65; Puchta et al. (1996), Proc. Natl. Acad. Sci. USA 93: 5055-60;Rong et al. (2002), Genes Dev. 16: 1568-81; Gouble et al. (2006), J.Gene Med. 8(5):616-622). Accordingly, attempts have been made toengineer meganucleases to exhibit novel binding specificity at medicallyor biotechnologically relevant sites (Porteus et al. (2005), Nat.Biotechnol. 23: 967-73; Sussman et al. (2004), J. Mol. Biol. 342: 31-41;Epinat et al. (2003), Nucleic Acids Res. 31: 2952-62; Chevalier et al.(2002) Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic Acids Res.31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques et al.(2007) Current Gene Therapy 7:49-66; U.S. Patent Publication Nos.20070117128; 20060206949; 20060153826; 20060078552; and 20040002092). Inaddition, naturally-occurring or engineered DNA-binding domains frommeganucleases have also been operably linked with a cleavage domain froma heterologous nuclease (e.g., FokI).

In other embodiments, the nuclease is a zinc finger nuclease (ZFN) orTALE DNA binding domain-nuclease fusion (TALEN). ZFNs and TALENscomprise a DNA binding domain (zinc finger protein or TALE DNA bindingdomain) that has been engineered to bind to a target site in a gene ofchoice and cleavage domain or a cleavage half-domain

As described in detail above, zinc finger binding domains and TALE DNAbinding domains can be engineered to bind to a sequence of choice. See,for example, Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo etal. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) NatureBiotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol.12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416. Anengineered zinc finger binding domain or TALE protein can have a novelbinding specificity, compared to a naturally-occurring protein.Engineering methods include, but are not limited to, rational design andvarious types of selection. Rational design includes, for example, usingdatabases comprising triplet (or quadruplet) nucleotide sequences andindividual zinc finger or TALE amino acid sequences, in which eachtriplet or quadruplet nucleotide sequence is associated with one or moreamino acid sequences of zinc fingers or TALE repeat units which bind theparticular triplet or quadruplet sequence. See, for example, co-ownedU.S. Pat. Nos. 6,453,242 and 6,534,261, incorporated by reference hereinin their entireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB2,338,237. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-owned WO02/077227.

Selection of target sites; and methods for design and construction offusion proteins (and polynucleotides encoding same) are known to thoseof skill in the art and described in detail in U.S. Patent PublicationNos. 20050064474 and 20060188987, incorporated by reference in theirentireties herein.

In addition, as disclosed in these and other references, zinc fingerdomains, TALEs and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. (e.g., TGEKP (SEQ IDNO:140), TGGQRP (SEQ ID NO:132), TGQKP (SEQ ID NO:133), and/or TGSQKP(SEQ ID NO:134)). See, e.g., U.S. Pat. Nos. 6,479,626; 6,903,185; and7,153,949 for exemplary linker sequences 6 or more amino acids inlength. The proteins described herein may include any combination ofsuitable linkers between the individual zinc fingers of the protein.See, also, U.S. Pat. No. 8,772,453.

Nucleases such as ZFNs, TALENs and/or meganucleases also comprise anuclease (cleavage domain, cleavage half-domain). As noted above, thecleavage domain may be heterologous to the DNA-binding domain, forexample a zinc finger DNA-binding domain and a cleavage domain from anuclease or a meganuclease DNA-binding domain and cleavage domain from adifferent nuclease. Heterologous cleavage domains can be obtained fromany endonuclease or exonuclease. Exemplary endonucleases from which acleavage domain can be derived include, but are not limited to,restriction endonucleases and homing endonucleases. See, for example,2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort etal. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes whichcleave DNA are known (e.g., 51 Nuclease; mung bean nuclease; pancreaticDNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn etal. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One ormore of these enzymes (or functional fragments thereof) can be used as asource of cleavage domains and cleavage half-domains.

Similarly, a cleavage half-domain can be derived from any nuclease orportion thereof, as set forth above, that requires dimerization forcleavage activity. In general, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains.Alternatively, a single protein comprising two cleavage half-domains canbe used. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof). In addition, the target sites for the two fusionproteins are preferably disposed, with respect to each other, such thatbinding of the two fusion proteins to their respective target sitesplaces the cleavage half-domains in a spatial orientation to each otherthat allows the cleavage half-domains to form a functional cleavagedomain, e.g., by dimerizing. Thus, in certain embodiments, the nearedges of the target sites are separated by 5-8 nucleotides or by 15-18nucleotides. However any integral number of nucleotides or nucleotidepairs can intervene between two target sites (e.g., from 2 to 50nucleotide pairs or more). In general, the site of cleavage lies betweenthe target sites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme Fok I catalyzesdouble-stranded cleavage of DNA, at 9 nucleotides from its recognitionsite on one strand and 13 nucleotides from its recognition site on theother. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768;Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al.(1994b) J. Biol. Chem. 269:31,978-31,982. Thus, in one embodiment,fusion proteins comprise the cleavage domain (or cleavage half-domain)from at least one Type IIS restriction enzyme and one or more zincfinger binding domains, which may or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is Fok I. This particular enzyme isactive as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA95: 10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the Fok I enzyme used in the disclosed fusionproteins is considered a cleavage half-domain. Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger-Fok I fusions, two fusion proteins, eachcomprising a FokI cleavage half-domain, can be used to reconstitute acatalytically active cleavage domain. Alternatively, a singlepolypeptide molecule containing a zinc finger binding domain and two FokI cleavage half-domains can also be used. Parameters for targetedcleavage and targeted sequence alteration using zinc finger-Fok Ifusions are provided elsewhere in this disclosure.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in InternationalPublication WO 07/014275, incorporated herein in its entirety.Additional restriction enzymes also contain separable binding andcleavage domains, and these are contemplated by the present disclosure.See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

In certain embodiments, the cleavage domain comprises one or moreengineered cleavage half-domain (also referred to as dimerization domainmutants) that minimize or prevent homodimerization, as described, forexample, in U.S. Patent Publication Nos. 20050064474, 20060188987 and20080131962, the disclosures of all of which are incorporated byreference in their entireties herein. Amino acid residues at positions446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531,534, 537, and 538 of Fok I are all targets for influencing dimerizationof the Fok I cleavage half-domains.

Exemplary engineered cleavage half-domains of Fok I that form obligateheterodimers include a pair in which a first cleavage half-domainincludes mutations at amino acid residues at positions 490 and 538 ofFok I and a second cleavage half-domain includes mutations at amino acidresidues 486 and 499.

Thus, in one embodiment, a mutation at 490 replaces Glu (E) with Lys(K); the mutation at 538 replaces Iso (I) with Lys (K); the mutation at486 replaced Gln (Q) with Glu (E); and the mutation at position 499replaces Iso (I) with Lys (K). Specifically, the engineered cleavagehalf-domains described herein were prepared by mutating positions 490(E→K) and 538 (I→K) in one cleavage half-domain to produce an engineeredcleavage half-domain designated “E490K:I538K” and by mutating positions486 (Q→E) and 499 (I→L) in another cleavage half-domain to produce anengineered cleavage half-domain designated “Q486E:I499L”. The engineeredcleavage half-domains described herein are obligate heterodimer mutantsin which aberrant cleavage is minimized or abolished. See, e.g., U.S.Patent Publication No. 2008/0131962, the disclosure of which isincorporated by reference in its entirety for all purposes. In certainembodiments, the engineered cleavage half-domain comprises mutations atpositions 486, 499 and 496 (numbered relative to wild-type FokI), forinstance mutations that replace the wild type Gln (Q) residue atposition 486 with a Glu (E) residue, the wild type Iso (I) residue atposition 499 with a Leu (L) residue and the wild-type Asn (N) residue atposition 496 with an Asp (D) or Glu (E) residue (also referred to as a“ELD” and “ELE” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490,538 and 537 (numbered relative to wild-type FokI), for instancemutations that replace the wild type Glu (E) residue at position 490with a Lys (K) residue, the wild type Iso (I) residue at position 538with a Lys (K) residue, and the wild-type His (H) residue at position537 with a Lys (K) residue or a Arg (R) residue (also referred to as“KKK” and “KKR” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490 and537 (numbered relative to wild-type FokI), for instance mutations thatreplace the wild type Glu (E) residue at position 490 with a Lys (K)residue and the wild-type His (H) residue at position 537 with a Lys (K)residue or a Arg (R) residue (also referred to as “KIK” and “KIR”domains, respectively). (See U.S. Patent Publication No. 20110201055).

Engineered cleavage half-domains described herein can be prepared usingany suitable method, for example, by site-directed mutagenesis ofwild-type cleavage half-domains (Fok I) as described in U.S. PatentPublication Nos. 20050064474 and 20080131962.

Alternatively, nucleases may be assembled in vivo at the nucleic acidtarget site using so-called “split-enzyme” technology (see e.g. U.S.Patent Publication No. 20090068164). Components of such split enzymesmay be expressed either on separate expression constructs, or can belinked in one open reading frame where the individual components areseparated, for example, by a self-cleaving 2A peptide or IRES sequence.Components may be individual zinc finger binding domains or domains of ameganuclease nucleic acid binding domain.

Nucleases (e.g., ZFNs and/or TALENs) can be screened for activity priorto use, for example in a yeast-based chromosomal system as described inWO 2009/042163 and U.S. Patent Publication No. 20090068164. Nucleaseexpression constructs can be readily designed using methods known in theart. See, e.g., United States Patent Publications 20030232410;20050208489; 20050026157; 20050064474; 20060188987; 20060063231; andInternational Publication WO 07/014275. Expression of the nuclease maybe under the control of a constitutive promoter or an induciblepromoter, for example the galactokinase promoter which is activated(de-repressed) in the presence of raffinose and/or galactose andrepressed in presence of glucose.

Delivery

The proteins (e.g., ZFPs, TALEs, ZFNs and/or TALENs), polynucleotidesencoding same and compositions comprising the proteins and/orpolynucleotides described herein may be delivered to a target cell byany suitable means, including, for example, by injection of the proteinor mRNA. Suitable cells include but not limited to eukaryotic andprokaryotic cells and/or cell lines. Non-limiting examples of such cellsor cell lines generated from such cells include T-cells, COS, CHO (e.g.,CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK,WI38, V79, B14AF28-G3, BHK, HaK, NS0, SP2/0-Ag14, HeLa, HEK293 (e.g.,HEK293-F, HEK293-H, HEK293-T), and perC6 cells as well as insect cellssuch as Spodoptera fugiperda (Sf), or fungal cells such asSaccharomyces, Pichia and Schizosaccharomyces. In certain embodiments,the cell line is a CHO-K1, MDCK or HEK293 cell line. Suitable cells alsoinclude stem cells such as, by way of example, embryonic stem cells,induced pluripotent stem cells (iPS cells), hematopoietic stem cells,neuronal stem cells and mesenchymal stem cells.

Methods of delivering proteins comprising DNA-binding domains asdescribed herein are described, for example, in U.S. Pat. Nos.6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558;6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, thedisclosures of all of which are incorporated by reference herein intheir entireties.

DNA binding domains and fusion proteins comprising these DNA bindingdomains as described herein may also be delivered using vectorscontaining sequences encoding one or more of the DNA-binding protein(s).Additionally, donor nucleic acids also may be delivered via thesevectors. Any vector systems may be used including, but not limited to,plasmid vectors, retroviral vectors, lentiviral vectors, adenovirusvectors, poxvirus vectors; herpesvirus vectors and adeno-associatedvirus vectors, etc. See, also, U.S. Pat. Nos. 6,534,261; 6,607,882;6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, incorporatedby reference herein in their entireties. Furthermore, it will beapparent that any of these vectors may comprise one or more DNA-bindingprotein-encoding sequences and/or donor nucleic acids as appropriate.Thus, when one or more DNA-binding proteins as described herein areintroduced into the cell, and donor DNAs as appropriate, they may becarried on the same vector or on different vectors. When multiplevectors are used, each vector may comprise a sequence encoding one ormultiple DNA-binding proteins and donor nucleic acids as desired.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding engineered DNA-binding proteins incells (e.g., mammalian cells) and target tissues and to co-introducedonors as desired. Such methods can also be used to administer nucleicacids encoding DNA-binding proteins to cells in vitro. In certainembodiments, nucleic acids encoding DNA-binding proteins s areadministered for in vivo or ex vivo gene therapy uses. Non-viral vectordelivery systems include DNA plasmids, naked nucleic acid, and nucleicacid complexed with a delivery vehicle such as a liposome or poloxamer.Viral vector delivery systems include DNA and RNA viruses, which haveeither episomal or integrated genomes after delivery to the cell. For areview of gene therapy procedures, see Anderson, Science 256:808-813(1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey,TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller,Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154(1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995);Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995);Haddada et al., in Current Topics in Microbiology and ImmunologyDoerfler and Böhm (eds.) (1995); and Yu et al., Gene Therapy 1:13-26(1994).

Methods of non-viral delivery of nucleic acids include electroporation,lipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Sonoporationusing, e.g., the Sonitron 2000 system (Rich-Mar) can also be used fordelivery of nucleic acids.

Additional exemplary nucleic acid delivery systems include thoseprovided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc.(Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) andCopernicus Therapeutics Inc, (see for example U.S. Pat. No. 6,008,336).Lipofection is described in e.g., U.S. Pat. No. 5,049,386, U.S. Pat. No.4,946,787; and U.S. Pat. No. 4,897,355) and lipofection reagents aresold commercially (e.g., Transfectam™ and Lipofectin™). Cationic andneutral lipids that are suitable for efficient receptor-recognitionlipofection of polynucleotides include those of Felgner, WO 91/17424, WO91/16024. Delivery can be to cells (ex vivo administration) or targettissues (in vivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

Additional methods of delivery include the use of packaging the nucleicacids to be delivered into EnGeneIC delivery vehicles (EDVs). These EDVsare specifically delivered to target tissues using bispecific antibodieswhere one arm of the antibody has specificity for the target tissue andthe other has specificity for the EDV. The antibody brings the EDVs tothe target cell surface and then the EDV is brought into the cell byendocytosis. Once in the cell, the contents are released (see MacDiarmidet al (2009) Nature Biotechnology vol 27(7) p. 643).

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding engineered DNA-binding proteins and donors as desiredtakes advantage of highly evolved processes for targeting a virus tospecific cells in the body and trafficking the viral payload to thenucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro and the modified cellsare administered to patients (ex vivo). Conventional viral based systemsfor the delivery of DNA-binding proteins and donors include, but are notlimited to, retroviral, lentivirus, adenoviral, adeno-associated,vaccinia and herpes simplex virus vectors for gene transfer. Integrationin the host genome is possible with the retrovirus, lentivirus, andadeno-associated virus gene transfer methods, often resulting in longterm expression of the inserted transgene. Additionally, hightransduction efficiencies have been observed in many different celltypes and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system depends on thetarget tissue. Retroviral vectors are comprised of cis-acting longterminal repeats with packaging capacity for up to 6-10 kb of foreignsequence. The minimum cis-acting LTRs are sufficient for replication andpackaging of the vectors, which are then used to integrate thetherapeutic gene into the target cell to provide permanent transgeneexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SIV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications in which transient expression is preferred, adenoviralbased systems can be used. Adenoviral based vectors are capable of veryhigh transduction efficiency in many cell types and do not require celldivision. With such vectors, high titer and high levels of expressionhave been obtained. This vector can be produced in large quantities in arelatively simple system. Adeno-associated virus (“AAV”) vectors arealso used to transduce cells with target nucleic acids, e.g., in the invitro production of nucleic acids and peptides, and for in vivo and exvivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47(1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994).Construction of recombinant AAV vectors are described in a number ofpublications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol.Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol.4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989).

At least six viral vector approaches are currently available for genetransfer in clinical trials, which utilize approaches that involvecomplementation of defective vectors by genes inserted into helper celllines to generate the transducing agent.

pLASN and MFG-S are examples of retroviral vectors that have been usedin clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn etal., Nat. Med. 1:1017-102 (1995); Malech et al., PNAS 94:22 12133-12138(1997)). PA317/pLASN was the first therapeutic vector used in a genetherapy trial. (Blaese et al., Science 270:475-480 (1995)). Transductionefficiencies of 50% or greater have been observed for MFG-S packagedvectors. (Ellem et al., Immunol Immunother. 44(1):10-20 (1997); Dranoffet al., Hum. Gene Ther. 1:111-2 (1997).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther.9:748-55 (1996)). Other AAV serotypes, including AAV1, AAV3, AAV4, AAV5,AAV6,AAV8, AAV9 and AAV10 can also be used in accordance with thepresent invention.

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer and readily infect a number of different celltypes. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including nondividing, differentiated cells such asthose found in liver, kidney and muscle. Conventional Ad vectors have alarge carrying capacity. An example of the use of an Ad vector in aclinical trial involved polynucleotide therapy for antitumorimmunization with intramuscular injection (Sterman et al., Hum. GeneTher. 7:1083-9 (1998)). Additional examples of the use of adenovirusvectors for gene transfer in clinical trials include Rosenecker et al.,Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:71083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarezet al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther.5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and w2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by a producer cellline that packages a nucleic acid vector into a viral particle. Thevectors typically contain the minimal viral sequences required forpackaging and subsequent integration into a host (if applicable), otherviral sequences being replaced by an expression cassette encoding theprotein to be expressed. The missing viral functions are supplied intrans by the packaging cell line. For example, AAV vectors used in genetherapy typically only possess inverted terminal repeat (ITR) sequencesfrom the AAV genome which are required for packaging and integrationinto the host genome. Viral DNA is packaged in a cell line, whichcontains a helper plasmid encoding the other AAV genes, namely rep andcap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. Accordingly, a viral vector can be modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the outer surface of the virus. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han et al., Proc. Natl. Acad.Sci. USA 92:9747-9751 (1995), reported that Moloney murine leukemiavirus can be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other virus-target cell pairs, in which the target cell expresses areceptor and the virus expresses a fusion protein comprising a ligandfor the cell-surface receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., FAB or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences which favor uptake byspecific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byre-implantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Ex vivo cell transfection for diagnostics, research, transplant or forgene therapy (e.g., via re-infusion of the transfected cells into thehost organism) is well known to those of skill in the art. In apreferred embodiment, cells are isolated from the subject organism,transfected with a DNA-binding proteins nucleic acid (gene or cDNA), andre-infused back into the subject organism (e.g., patient). Various celltypes suitable for ex vivo transfection are well known to those of skillin the art (see, e.g., Freshney et al., Culture of Animal Cells, AManual of Basic Technique (3rd ed. 1994)) and the references citedtherein for a discussion of how to isolate and culture cells frompatients).

In one embodiment, stem cells are used in ex vivo procedures for celltransfection and gene therapy. The advantage to using stem cells is thatthey can be differentiated into other cell types in vitro, or can beintroduced into a mammal (such as the donor of the cells) where theywill engraft in the bone marrow. Methods for differentiating CD34+ cellsin vitro into clinically important immune cell types using cytokinessuch a GM-CSF, IFN-γ and TNF-α are known (see Inaba et al., J. Exp. Med.176:1693-1702 (1992)).

Stem cells are isolated for transduction and differentiation using knownmethods. For example, stem cells are isolated from bone marrow cells bypanning the bone marrow cells with antibodies which bind unwanted cells,such as CD4+ and CD8+(T cells), CD45+(panB cells), GR-1 (granulocytes),and Tad (differentiated antigen presenting cells) (see Inaba et al., J.Exp. Med. 176:1693-1702 (1992)).

Stem cells that have been modified may also be used in some embodiments.For example, neuronal stem cells that have been made resistant toapoptosis may be used as therapeutic compositions where the stem cellsalso contain the ZFP TFs of the invention. Resistance to apoptosis maycome about, for example, by knocking out BAX and/or BAK using BAX- orBAK-specific ZFNs (see, U.S. Pat. No. 8,597,912) in the stem cells, orthose that are disrupted in a caspase, again using caspase-6 specificZFNs for example. These cells can be transfected with the ZFP TFs thatare known to regulate HLA.

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic DNA-binding proteins (or nucleic acids encoding theseproteins) can also be administered directly to an organism fortransduction of cells in vivo. Alternatively, naked DNA can beadministered. Administration is by any of the routes normally used forintroducing a molecule into ultimate contact with blood or tissue cellsincluding, but not limited to, injection, infusion, topical applicationand electroporation. Suitable methods of administering such nucleicacids are available and well known to those of skill in the art, and,although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route.

Methods for introduction of DNA into hematopoietic stem cells aredisclosed, for example, in U.S. Pat. No. 5,928,638. Vectors useful forintroduction of transgenes into hematopoietic stem cells, e.g., CD34⁺cells, include adenovirus Type 35.

Vectors suitable for introduction of transgenes into immune cells (e.g.,T-cells) include non-integrating lentivirus vectors. See, for example,Ory et al. (1996) Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull et al.(1998) J. Virol. 72:8463-8471; Zuffery et al. (1998) J. Virol.72:9873-9880; Follenzi et al. (2000) Nature Genetics 25:217-222.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositionsavailable, as described below (see, e.g., Remington's PharmaceuticalSciences, 17th ed., 1989).

As noted above, the disclosed methods and compositions can be used inany type of cell including, but not limited to, prokaryotic cells,fungal cells, Archaeal cells, plant cells, insect cells, animal cells,vertebrate cells, mammalian cells and human cells, including T-cells andstem cells of any type. Suitable cell lines for protein expression areknown to those of skill in the art and include, but are not limited toCOS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11), VERO, MDCK, WI38,V79, B14AF28-G3, BHK, HaK, NS0, SP2/0-Ag14, HeLa, HEK293 (e.g.,HEK293-F, HEK293-H, HEK293-T), perC6, insect cells such as Spodopterafugiperda (Sf), and fungal cells such as Saccharomyces, Pichia andSchizosaccharomyces. Progeny, variants and derivatives of these celllines can also be used.

Applications

The disclosed compositions and methods can be used for any applicationin which it is desired to modulate HLA genes and/or HLA regulators. Inparticular, these methods and compositions can be used where modulationor modification of a HLA allele is desired, including but not limitedto, therapeutic and research applications.

Diseases and conditions which are tied to HLA include Addison's disease,ankylosing spondylitis, Behçet's disease, Buerger's disease, celiacdisease, chronic active hepatitis, Graves' disease, juvenile rheumatoidarthritis, psoriasis, psoriatic arthritis, rheumatoid arthritis, Sjögrensyndrome, and lupus erythematosus, among others. In addition,modification of a HLA gene may be useful in conjunction with othergenetic modifications of a cell of interest. For example, modificationof a target cell such as a CTL with a chimeric antigen receptor tochange the CTL's specificity may be combined with HLA modification exvivo in order to develop a cell therapeutic that may be used in most anypatient in need thereof.

In addition, the materials and methods of the invention can be used inthe treatment, prevention or amelioration of graft-versus-host-disease.Graft-versus-host disease (GVHD) is a common complication when allogenicT-cells (e.g., bone marrow and/or blood transfusion) are administered toa patient. The functional immune cells in the infused material recognizethe recipient as “foreign” and mount an immunologic attack. Bymodulating HLA and/or TCR expression in allogenic T cells, “off theshelf” T cells (e.g., CD19-specific T-cells) can be administered ondemand as “drugs” because the risk of GVHD is reduced or eliminated.

Methods and compositions also include stem cell compositions wherein acopy of a HLA allele within the stem cells has been modified using aHLA-specific or HLA regulator specific ZFN. For example, HLA modifiedhematopoietic stem cells can be introduced into a patient following bonemarrow ablation. These altered HSC would allow the re-colonization ofthe patient without loss of the graft due to rejection. The introducedcells may also have other alterations to help during subsequent therapy(e.g., chemotherapy resistance) to treat the underlying disease.

The methods and compositions of the invention are also useful for thedevelopment of HLA modified platelets, for example for use astherapeutics. Thus, HLA modified platelets may be used to treatthrombocytopenic disorders such as idiopathic thrombocytopenic purpura,thrombotic thrombocytopenic purpura and drug-induced thrombocytopenicpurpura (e.g. heparin-induced thrombocytopenia). Other plateletdisorders that may be treated with the HLA modified platelets of theinvention include Gaucher's disease, aplastic anemia, Onyalai,fetomaternal alloimmune thrombocytopenia, HELLP syndrome, cancer andside effects from some chemotherapeutic agents. The HLA modifiedplatelets also have use in as an “off the shelf” therapy in emergencyroom situations with trauma patients.

The methods and compositions of the invention can be used inxenotransplantation. Specifically, by way of example only, pig organscan be used for transplantation into humans wherein the porcine MHCgenes have been deleted and/or replaced with human HLA genes. Strains ofpigs can be developed (from pig embryos that have had HLA targeting ZFNsencoded by mRNAs injected into them such that the endogenous MHC genesare disrupted, or from somatic cell nuclear transfer into pig embryosusing nuclei of cells that have been successfully had their HLA genestargeted) that contain these useful genetic mutations, and these animalsmay be grown for eventual organ harvest. This will prevent rejection ofthese organs in humans and increase the chances for successfultransplantation.

The methods and compositions of the invention are also useful for thedesign and implementation of in vitro and in vivo models, for example,animal models of HLA or other disorders, which allows for the study ofthese disorders.

EXAMPLES Example 1: Design, Construction and General Characterization ofZinc Finger Protein Nucleases (ZFN)

Zinc finger proteins were designed and incorporated into plasmids oradenoviral vectors essentially as described in Urnov et al. (2005)Nature 435(7042):646-651, Perez et al (2008) Nature Biotechnology26(7):808-816, and as described in U.S. Pat. No. 6,534,261. In addition,see U.S. Patent Publication No. 20110158957 for ZFNs targeted to TRACand TRBC. Table 1 shows the recognition helices within the DNA bindingdomain of exemplary ZFPs while Table 2 shows the target sites for theseZFPs. Nucleotides in the target site that are contacted by the ZFPrecognition helices are indicated in uppercase letters; non-contactednucleotides indicated in lowercase.

Example 2: ZFNs Specific for HLA Class I Genes

The HLA complexes often contain several family members co-localizedwithin the same general area of the genome. FIG. 1 presents a schematicof the arrangement of the major genes in the HLA class I and class Iloci found on chromosome 6.

ZFNs Directed Against the HLA A Locus

ZFN pairs were made to target the HLA A locus, with some madespecifically against the A2 allele while others were made to target theA3 locus or both. For the A2 and A3 alleles, two pairs were tested suchthat ZFN 18889 was paired with 18881, and 18889 was also paired with24859. When the successful pairing of the ZFNs creates a DSB at thedesired location, the site is often repaired using non-homologous endjoining (NHEJ). This process frequently results in the insertion ordeletion of a small number of nucleotides at the site of the mendedjunction, and so when the DNA around the junction is amplified by PCR,and then subjected to a Cel-I mismatch assay as described, for example,in U.S. Patent Publication Nos. 20080015164; 20080131962 and20080159996, (Surveyor™, Transgenomic), using the products amplifiedwith respective primers, the frequency of these insertions or deletions(collectively “indels”) can be calculated when the products of the assayare subjected to gel electrophoresis. Thus, the A2/A3 specific pairswere examined for activity against their targets using the Cel-I assay.Cells as indicated were transfected with GFP control or each of thepairs of ZFNs. DNA was prepared from the cells one day posttransfection, and the results are shown in FIG. 2. Arrows indicatecleavage was found only in samples containing ZFN pairs, but was notfound in the control samples wherein cells were transfected with ZFNsspecific for GFP. The percent gene modification is shown at the bottomof each lane.

The 18889/18881 pair gave approximately 3% gene modification while the18889/24859 pair gave approximately 6% NHEJ against the HLA A2 gene whenplasmids encoding the ZFNs were transfected into HEK293 cells. For theA3 allele, the 18889/18881 pair gave 9% and the 18889/24859 pair gave10% NHEJ activity. Since these pairs are capable of cutting both the A2and A3 alleles, it is possible to create a A2/A3 double knock-out cellline.

The presence of the HLA A marker on the cell surface was analyzed bystandard FACS analysis. Briefly, A2, A3 or A2,A3 disrupted HEK293 cellswere stained. HLA-A2 staining was done by anti-HLA-A2 PE (BDBioSciences, clone BB7.2). Mouse IgG2bk PE (BD BioSciences) was used forisotype control. For HLA-A3, we first stained these cells withbiotinylated anti-HLA-A3 Ab (Abcam, clone 4i53) and then SA-PE (BD). Thenegative controls in each experiment were: HLA-A2-igG2bK PE,HLA-A3-SA-PE staining without HLA-A3 Ab.

In these experiments, a positive control was performed using the isotypecontrol antibody described above where the results are seen in thefigures as a black line that is constant in all samples. The cultureswere then either stimulated for HLA expression by addition of IFN gamma(600 IU/mL)+TNF (10 mg/mL) for 48 hours, or used without stimulation.

As shown in FIG. 3, the lines closest to the isotype control peak arethe samples lacking stimulation, while the shifted peaks are those inthe presence of the IFN γ and TNF (see Figure legend). The set offigures on the left hand column are all probed with the anti-HLA A2antibody, while those on the right hand column were probed with theanti-HLA A3 antibody. As shown, the HLA markers as indicated are nolonger detectable when the corresponding HLA genes have beenfunctionally disrupted.

Next, the HLA A knock out HEK293 cell lines were analyzed to see if theycould be lysed by HLA-A restricted CTL cell lines. The methodology forthese experiments was as follows. Target cells were labeled with 0.1 mCiof ⁵¹Cr for 2 hours. After washing with ice-cold RPMI1640 supplementedwith 10% FBS thrice, labeled cells were diluted and distributed at 1×10³target cells/100 μL per well in 96-well, v-bottomed plates. After 30minutes incubation at room temperature with 10-fold serial dilutions ofthe peptides, CTL were added at indicated effector target ratio. After 4hr incubation at 37° C., 5% CO2 incubator, 50 μl of supernatants werecollected and count on TopCount (Perkin Elmer). All assays wereperformed in triplicate. Parental HEK293 cell lines and HLA knocked downHEK293 clones were treated with 600 IU/mL of interferon-γ (IFN-γ; R&DSystems®) and 10 ng/mL of tissue necrosis factor-α (TNF-α; R&D Systems®)for 48 hours before assay. The percent specific lysis was calculated asfollows: ((experimental cpm−spontaneous cpm)/(maximum cpm−spontaneouscpm))×100. In these examples, the peptide antigen target was added infor display by any functioning HLA class I complexes, and in thepresence of a functioning HLA A-peptide complex, the CTL clones are ableto attack the cells and cause lysis.

As shown in FIG. 4, the HEK293 clones lacking the A2 or the A3 HLAmakers were resistant to lysis induced by either the 7A7 PANE1/A3 CTLclone (panel A) or the GAS2B3-5 C19ORF48/A2 CTL clone (panel B) in thepresence of their cognate peptide antigens.

ZFN mediated HLA k.o. was repeated in primary T cells. mRNAs encodingthe 18889 (containing the KKR FokI variation) and 24859 (containing theELD FokI variation) ZFNs were nucleofected into primary T cells of ahomozygous HLA A2 genotype as follows. 5×10⁶ primary T cells (isolatedby standard methods) were nucleofected using an Amaxa Nucleofector®system (program T20) with the ZFN encoding mRNAs using 2.5-10 μg eachmRNA per reaction in 100 μL of buffer as supplied by the manufacturer(Lonza). These cells were then analyzed by FACs analysis to determine ifthe HLA A2 markers were present on the cell surface by standardmethodology.

As shown in FIG. 5, the percent of cells lacking HLA-A expression rangedfrom approximately 19-42% following this treatment. FIG. 5A shows thepercent of cells lacking HLA-A2 under standard treatment conditions,while FIG. 5B shows the percent of cells lacking HLA-A2 using the“transient cold shock” treatment conditions (see co-owned U.S. Pat. No.8,772,008).

In addition, we verified that the loss of HLA A2 expression was causedby ZFN mediated modification of the HLA A2 gene by Cel I analysis asdescribed previously. FIG. 6 shows the Cel I data where the determinedpercent NHEJ activity at the ZFN target site ranged from 3-28%. In thisfigure, “wt” refers to a wild type FokI domain, while “mut” refers tothe EL/KK FokI mutant pair described above. These data demonstrate thatthe methods and compositions of the invention can be used to delete HLAA expression and create HLA A null cell lines and primary T cells.

HLA A knock out cells can be enriched to increase the percent HLA A nullcells present. A ZFN treated population of T cells, where some lowpercent of the cells were HLA A null were treated with anti-HLA A2antibodies tagged with phycoerythrin (PE) (BD BioSciences clone 7.2).Next, beads tagged with an anti-PE antibodies (Miltenyi) were used tobind and thus separate the cells expressing HLA A2 from the HLA A2 nullcells according to the manufacturer's directions. Using this technique,the cell populations went from a range of 5.1-34% HLA Asnull cells asassayed by FACs analysis to a range of 92-95% HLA A2 null.

ZFNs Directed to the HLA B and C Loci

ZFNs were constructed to target the HLA B and C loci and tested usingthe Cel I assay as described above.

As shown in FIG. 7, these ZFN were successfully able to induce genemodification within both of these genes (HLA C knock out is shown inpanel A using ZFNs 25588/25589 while the HLA B knock out in panel B wasmade using the ZFN pair 25316/25317). These targets are located withinthe gene sequences, so can be used to create HLA B and HLA C knock outclones.

Large Deletions of MHC Class I Complex

ZFNs were also designed to allow for a large deletion with the HLA classI locus which simultaneously deletes the HLA B and HLA C genes. Thesesets of ZFNs were designed to cut upstream of the HLA B gene (HLA B-up)and downstream of the HLA C gene (HLA C-down). These ZFN pairs weretested individually using the Cel I assay as described above to see thedegree of cutting as assayed by NHEJ activity.

FIG. 8A shows the HLA C-down results for pair 15296/15298 for both wtFokI domains (wt) and the EL/KK Fok I domain (mut). FIG. 8B shows asimilar set of data for the HLA B-up pairs 15267/15265 and 17454/17456in K562 cells. Percent gene modification is indicated at the bottom ofthe lanes (“% NHEJ”). These ZFNs were then used to test if the HLA B andHLA C genes could be deleted by a combination of these two sets of ZFNs.FIG. 9 shows a diagram of the assay used (panel A), and also shows theresults of the PCR (panel B). K562 cells were transfected with the twosets of ZFNs and DNA isolated from the cells 3 or 10 days aftertransfection as indicated.

PCR was performed using primers flanking the region between the HLA-Band HLA-C as illustrated in FIG. 9A. Because of the large amount of DNAto be deleted by a successful double deletion (approximately 100 Kb),the PCR reaction is only successful when the deletion has been made.FIG. 9B shows a gel with a PCR reaction following treatment with the twoZFN pairs. The left side of the gel shows a dilution series of a plasmidused for rough quantitation of the amount of deletion PCR productpresent on the right side of the gel. In this figure, “wt” refers to awild type FokI domain, while “mut” refers to the EL/KK FokI mutant pair.The results show that approximately 5% of the DNA present contained thedeletion.

Clones derived from this experiment were subjected to FACs analysis toobserve the expression of the class I complex overall, and specificallythe HLA B and C genes in particular. This was done as described aboveusing an anti-class I antibody to analyze class I expression, and ananti-HLA BC antibody to analyze HLA BC. The results showed that in theparental K562 cell line, 9.02% of the cells expressed the class Icomplex in general, and 15.98% of the cells expressed HLA B and C. Inone knock out line, the class I expression level was 3.88% and HLA BCexpression was 7.82%. It is likely that this line does not contain aknock out on both HLA BC alleles, and so it is not unexpected that theexpression would be only reduced rather than eliminated.

Example 3: ZFNs Specific for HLA Class I Regulator Genes

In addition to examining the action of ZFNs specific for class I HLAgenes, we also looked at the effects of ZFNs directed to potential classI regulators, namely TAP1, TAP2 and tapasin. ZFNs were made againstthese gene targets, and their design details are shown in Tables 1 and2.

The ZFNs were transfected into HEK293 cells and tested for genemodification activity by the Cel-I assay as described above

As shown in FIGS. 10 and 11, ZFN pairs modified their targets. FIG. 10 Ashows the results of the ZFNs specific for TAP1 (pair 28386/28385) wherethe data indicates an approximate 39% gene modification activity. FIG.10B shows that the ZFN pair 28394/28393 modifies Tap2 with about 49%efficiency. FIG. 11 shows similar results for ZFNs specific for theTapasin gene (pair 28406/28405 and pair 28404/28403), where the dataindicates an approximate 34 and 64% gene modification activity,respectively.

Example 4: ZFNs Specific for HLA Class II Genes

As described above in Example 2 for the class I genes, large deletionswere also made in the class II gene cluster. Two ZFN pairs wereidentified that cleave the target DNA upstream of the DBP2 gene (15872and 15873) and downstream of the DRA gene (15909 and 15910),respectively. Each pair was analyzed by Cel I analysis in K562 cells asdescribed above. The Cel I analysis displayed in FIG. 12 shows that forthe 15872/15873 pair, 13% NHEJ was found using the ZFN versionscontaining the wild type (wt) FokI domains, while when the pair was madewith the EL/KK FokI pair (mut) as described previously, the NHEJactivity was found to be approximately 28%. For the 15909/15910 pair, 6and 11% NHEJ activity was found using the ZFN versions containing thewild type (wt) FokI domains, while 14% NHEJ activity was observed withthe EL/KK FokI domain pair (mut).

The two ZFN pairs were used together to delete the section of DNAbetween DBP2 and DRA and then a PCR with primers flanking the deletionwas performed as described above in Example 2. The PCR products wereanalyzed and compared with a dilution series to estimate the percent ofdeletion present. As shown in FIG. 13, approximately 0.04% of thealleles present showed evidence of the deletion.

The junction across the joined sections was sequenced and results areshown in FIG. 14. Line 6 shows the genomic reference sequence at thetarget site of ZFN 15873 upstream of DBP2 (with the ZFN binding siteitself underlined). Line 5 shows the genomic reference sequence aroundthe binding site of ZFN 15909 downstream of DRA (with the ZFN bindingsite itself underlined). Lines 1-4 show the sequence of 4 separatesubclones of the PCR product as described above and demonstrate thatboth target sequences are present indicating that the two ends havejoined at the ZFN cleavage sites following the deletion. Line 7 showsthe consensus of the deletion products.

These results indicate that large deletions (approximately 700 Kb) canbe made to delete portions of the HLA class II complex.

Example 5: ZFNs Specific for HLA Class II Regulator Genes

As described previously, the class II complex appears to be regulated bya master regulatory molecule CIITA. Thus, if the CIITA gene weredisrupted or manipulated, it might be possible to disrupt or alter HLAclass II expression as a whole. Thus, ZFN pairs were made to target theCIITA gene. Additionally, the RFX5 gene product appears to be part ofthe class II enhanceosome, and so disruption of this gene may alsodisrupt or alter HLA class II expression. Accordingly, ZFN pairstargeting this gene were made and tested as well. Both sets of ZFNs weretested using the Cel I assay as described above in K562 cells.

As shown in FIG. 15, Cel I mismatch assay results show that the CIITAtargeted ZFN pair (15486/15487) was able to cause gene modification inapproximately 15% of the alleles, while the RFX5 targeted ZFN pair(15506/15507) caused gene modification in about 2% of the alleles.Control reactions include a mock transfection with no added DNA and atransfection using a GFP expression plasmid.

Next, the CIITA targeted ZFN pairs were tested by transfecting into RAJIcells. These cells are a lymphoblastoid cell line that is known toexpress HLA class II.

The gel depicted in FIG. 16 shows a comparison of the Cel I activity inK562 cells alongside RAJI cells. ‘K’ depicts the results in K562 cellsand ‘R’ depicts the results in RAJI cells. ‘n.c.’ depicts the negativecontrol in cells without any added ZFN DNA during transfection. Theresults demonstrate that in K562 cells, there was approximately 12-15%gene modification activity observed and in RAJI cells, approximately 1%probably reflecting the poorer transfection efficiency in RAJI cells.

Example 6: Use of HLA Knock Out Cells in Combination with AnotherGenetic Modification

The CD19 marker is a cell surface marker that is expressed on 95% of allB-cell malignancies. It is not expressed on hematopoietic stem cells, oron normal tissues outside the B lineage; and is lost upondifferentiation of B cells to mature plasma cells. Thus, CD19 representsan attractive target for targeted immunotherapy for treatment of B celllymphomas and B-ALL cells. T-cells containing a chimeric antigenreceptor (CAR) specific for CD19 have been created (see Davies et al(2010) Cancer Res 70(10):3915-24) through transposon aided genomicinsertion. Deletion of the HLA markers could allow such a cell therapyproduct to be used for a number of patients rather than just those withthe matching HLA haplotype. Thus, the CAR-19 modified T cells weretreated with the HLA A specific ZFNs as described above in Example 2.The cells were then analyzed by FACS analysis as described above.

As shown in FIG. 17, analyzing the cells using a negative (isotype)control antibody (mouse anti-IgG2 (BD BioSciences)) showed a 0.3%negative (non-specific binding) signal for HLA-A2 (as described above)expression. When mock transfected cells were analyzed with the HLA-A2antibody (no mRNA), 1.6% of the cells did not expressing HLA-A2. A bulkpopulation of T cells treated with ZFNs showed that 17.6% of the cellswere HLA A2 null, but following enrichment for HLA A2 non-expressers asdescribed above in Example 2, 95.3% of the cells were HLA A2 null(compare “Enriched” with “HLA-A.ZFN bulk”) in FIG. 17.

The HLA A2 null T cells were then treated with HLA A2-specific CTLs. Asis shown in FIG. 18, the cells that were HLA A2 null were resistant tolysis by the CTLs. These experiments were carried out as describedpreviously. The CTLs used were the GAS2B3-5 C19ORF48/A2 cells(CIPPDSLLFPA (SEQ ID NO:136) epitope) described previously (see Example2) and are specific for HLA A2.

These data demonstrate that ZFP mediated HLA knockouts can be made incells that carry another useful genetic modification and thus allow awider use of these therapeutics.

Example 7: TCR Knock Out

Use of the CAR-19 modified T cells as described above in Example 6 couldbe potentially hampered in an allogenic setting due to endogenous TCRαβexpression. Thus, ZFN reagents designed to disrupt either the TCRα orthe TCRβ constant chains were tested in primary T cells. The ZFN pair25539/25540 was used for the TCRα knockout and the ZFN pair 16783/16787was used for the TCRβ knockout. In these experiments, 1 million primaryT cells were subjected to nucleofection with the Amaxa system using theZFN encoding mRNAs as described above. Cells were then subjected to bothFACs and Cel I analyses.

As shown in FIG. 19, cells lacking CD3 expression increase from 2.4%,without any nucleofected TCR ZFN mRNAs, to 9.4% in the presence of 10 μgTCRβ-specific ZFN mRNA. In the presence of 10 μg TCRα-specific ZNF mRNA,the percent of CD3 negative cells increases to 28.1%. The FACs data ispresented with the TRCβ data is shown across the top (“TRBC targetZFNs”) and the TCRα data is across the bottom (“TRAC target ZFNs”). Lackof expression of either the TCRα or the TCRβ chains is assayed by thepresence of the CD3 complex, in which a functioning TCR is required forstable presentation on the cell surface.

FIG. 20 depicts a gel with the results of the Cel I analysis, performedas described above on samples incubated under the transient hypothermicor the standard conditions, and the percent gene modification activitydata (displayed at the bottom of each lane) agrees roughly with the FACsanalysis that was done on cells incubated at 37 degrees.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entirety.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity of understanding,it will be apparent to those skilled in the art that various changes andmodifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing descriptions andexamples should not be construed as limiting.

What is claimed is:
 1. A method of making a genetically modifiedmammalian cell comprising an endogenous human leukocyte antigen (HLA)class I gene comprising a sequence selected from the group consisting ofany one of the nucleotides of SEQ ID NOs:97 to 111 and an endogenous HLAclass II gene comprising a sequence selected from the group consistingof any one of the nucleotides of SEQ ID NOs:112 to 115, the geneticallymodified cell comprising: a first and second genomic modifications,wherein the first genetic modification comprises an integrated exogenousnucleotide sequence encoding a chimeric antigen receptor (CAR) or T-cellreceptor (TCR) gene and the second genomic modification comprises aninactivated HLA gene, the method comprising: (a) integrating the CAR orTCR gene into the cell; and (b) expressing a pair of zinc fingernucleases in the cell, each zinc finger nuclease comprising a cleavagedomain and the recognition helix regions of the zinc finger proteinsdesignated 18889, which binds to a target site within SEQ ID NO:97;11881, which binds to a target site within SEQ ID NO:98; 24859, whichbinds to a target site within SEQ ID NO:99; 25191, which binds to atarget site within SEQ ID NO: 100; 25190, which binds to a target sitewithin SEQ ID NO: 101; 25316, which binds to a target site within SEQ IDNO: 102; 25317, which binds to a target site within SEQ ID NO: 103;15267, which binds to a target site within SEQ ID NO: 103; 15265, whichbinds to a target site within SEQ ID NO: 104; 17454, which binds to atarget site within SEQ ID NO: 106; 17456, which binds to a target sitewithin SEQ ID NO: 107; 15296, which binds to a target site within SEQ IDNO: 108; 15298, which binds to a target site within SEQ ID NO: 109;25588, which binds to a target site within SEQ ID NO: 110; 25589, whichbinds to a target site within SEQ ID NO: 111; 15872, which binds to atarget site within SEQ ID NO: 112; 15873, which binds to a target sitewithin SEQ ID NO: 113; 15909, which binds to a target site within SEQ IDNO: 114; or 15910, which binds to a target site within SEQ ID NO: 115,wherein the pair of zinc finger nucleases includes 18889 and 18881;18889 and 24859; 25191 and 25190; 25316 and 25317; 15267 and 15265;17454 and 17456; 15296 and 15298; 25588 and 25589; 15872 and 15873; or15909 and 15910; and wherein the pair of zinc finger nuclease cleave andinactivate the endogenous HLA class I gene and/or HLA class II gene inthe mammalian cell.
 2. The method of claim 1, further comprising thestep of cleaving additional gene sites in the mammalian cell usingadditional pairs of zinc-finger proteins.
 3. The method of claim 2,wherein the additional gene is a safe harbor gene.
 4. The method ofclaim 3, wherein an exogenous sequence is integrated into the safeharbor gene.
 5. The method of claim 4, wherein the exogenous sequenceencodes a CAR.
 6. The method of claim 4, wherein the exogenous sequencecomprises a TCR gene.
 7. The method of claim 6, further wherein one ormore endogenous TCR genes are inactivated in the cell.
 8. The method ofclaim 7, wherein the cell is a T-cell or a stem cell.
 9. The method ofclaim 8, wherein the stem cell is selected from the group consisting ofan induced pluripotent stem cell (iPSC), a human embryonic stem cell(hES), a mesenchymal stem cell (MSC) or a neuronal stem cell.